EP4007591A1 - Matières et procédés pour le biotransport multidirectionnel - Google Patents

Matières et procédés pour le biotransport multidirectionnel

Info

Publication number
EP4007591A1
EP4007591A1 EP20849929.3A EP20849929A EP4007591A1 EP 4007591 A1 EP4007591 A1 EP 4007591A1 EP 20849929 A EP20849929 A EP 20849929A EP 4007591 A1 EP4007591 A1 EP 4007591A1
Authority
EP
European Patent Office
Prior art keywords
seq
sequence
cdr1
single domain
cdr2
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20849929.3A
Other languages
German (de)
English (en)
Other versions
EP4007591A4 (fr
Inventor
Rajkumar Ganesan
Bharathikumar Vellalore MARUTHACHALAM
Adam ZWOLAK
Brian GEIST
Xiefan Lin-Schmidt
Sathyadevi VENKATARAMANI
Sanjaya Singh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Biotech Inc
Original Assignee
Janssen Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=74502941&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=EP4007591(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Janssen Biotech Inc filed Critical Janssen Biotech Inc
Publication of EP4007591A1 publication Critical patent/EP4007591A1/fr
Publication of EP4007591A4 publication Critical patent/EP4007591A4/fr
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22Hormones
    • A61K38/26Glucagons
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22Hormones
    • A61K38/28Insulins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00Drugs for disorders of the respiratory system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/54Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541Mucosal route
    • A61K2039/544Mucosal route to the airways
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52Constant or Fc region; Isotype
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565Complementarity determining region [CDR]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567Framework region [FR]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/90Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/90Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • FIELD Provided herein are single domain antibodies (e.g., VHH domains) and uses thereof for delivering agents (e.g., therapeutic agents), including by transporting the agents from an apical surface of a polymeric immunoglobulin receptor (pIgR)-expressing cell to a basolateral surface of the pIgR-expressing cell.
  • agents e.g., therapeutic agents
  • pIgR polymeric immunoglobulin receptor
  • Biologics have been the driving force in pharmaceutical space with increasing potential to address many diseases, disorders, and conditions, including chronic diseases and various unmet medical needs. Indeed, the number of biologics in development continues to increase exponentially, particularly in the therapeutic areas of cancer and cancer related conditions, rare diseases, neurologic disorders, and immunological or inflammatory diseases, disorders, and conditions, including autoimmune disorders.
  • a method for delivering from an apical surface of a polymeric immunoglobulin receptor (pIgR)-expressing cell to a basolateral surface of the pIgR- expressing cell comprising contacting the pIgR-expressing cell with (i) a single domain antibody that binds to pIgR, or (ii) a therapeutic molecule comprising an agent and the single domain antibody.
  • pIgR polymeric immunoglobulin receptor
  • a method for transporting a therapeutic molecule to a basolateral surface of the pIgR-expressing cell of a subject comprising administering to the subject the therapeutic molecule comprising an agent and a single domain antibody.
  • the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the therapeutic agent is transported from an apical surface of a pIgR-expressing cell to a basolateral surface of the pIgR- expressing cell in the subject.
  • a method for transporting a therapeutic molecule to systemic circulation of a subject comprising administering to the subject the therapeutic molecule comprising an agent and a single domain antibody, wherein the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the therapeutic agent is transported from an apical surface of a pIgR-expressing cell to a basolateral surface of the pIgR-expressing cell in the subject.
  • a method for transporting a therapeutic molecule to lamina intestinal or gastrointestinal tract of a subject comprising administering to the subject the therapeutic molecule comprising an agent and a single domain antibody, wherein the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the therapeutic agent is transported from an apical surface of a pIgR-expressing cell to a basolateral surface of the pIgR-expressing cell in the subject.
  • the single domain antibody or the therapeutic molecule comprising the agent and the single domain antibody is capable of being transported from the basolateral surface of the pIgR-expressing cell to the apical surface of the pIgR-expressing cell.
  • the pIgR-expressing cell is an epithelial cell.
  • the epithelia cell is an intestinal lumen cell or an airway epithelial cell.
  • the agent is a diabetes medication.
  • the diabetes medication is selected from a group consisting of insulin, glucagon-like-peptide-1, insulin-mimic peptides, and glucagon-like-peptide-1-mimic peptides.
  • the agent is a peptide or an antibody or a fragment thereof.
  • the antibody or fragment thereof is selected from a group consisting of an anti-TNF-alpha antibody or a fragment thereof, an anti-IL23 antibody or a fragment thereof, and an antibody that binds to a receptor of IL23 or a fragment thereof.
  • the agent is a vaccine.
  • the vaccine is for preventing an infection selected from a group consisting of Vibrio, Cholera, Typhoid, Rotavirus, Tuberculosis, HIV, Flu, Ebola, and Sendai.
  • an infection selected from a group consisting of Vibrio, Cholera, Typhoid, Rotavirus, Tuberculosis, HIV, Flu, Ebola, and Sendai.
  • pIgR polymeric immunoglobulin receptor
  • the molecule is capable of being provided to a basolateral surface of an pIgR-expressing cell from an apical surface of the pIgR-expressing cell in the subject. [0015] In some embodiments, the molecule is capable of being provided to an apical surface of the pIgR-expressing cell from a basolateral surface of an pIgR-expressing cell in the subject. [0016] In some embodiments, the pIgR-expressing cell is an epithelial cell. In some embodiments, the epithelia cell is an intestinal lumen cell or an airway epithelial cell. [0017] In some embodiments, the agent is a diabetes medication.
  • the diabetes medication is selected from a group consisting of insulin, glucagon-like-peptide-1, insulin-mimic peptides, and glucagon-like-peptide-1-mimic peptides.
  • the agent is a peptide or an antibody or a fragment thereof.
  • the antibody or fragment thereof is selected from a group consisting of an anti-TNF-alpha antibody or a fragment thereof, an anti-IL23 antibody or a fragment thereof, and an antibody that binds to a receptor of IL23 or a fragment thereof.
  • the agent is a vaccine.
  • the vaccine is for preventing an infection selected from a group consisting of Vibrio, Cholera, Typhoid, Rotavirus, Tuberculosis, HIV, Flu, Ebola, and Sendai.
  • an infection selected from a group consisting of Vibrio, Cholera, Typhoid, Rotavirus, Tuberculosis, HIV, Flu, Ebola, and Sendai.
  • the molecule comprises an agent and a single domain antibody that binds to pIgR.
  • the agent is an antibody or fragment thereof, a peptide, a vaccine, a small molecule, a polynucleotide, a radioisotope, a toxin, an enzyme, an anticoagulant, a hormone, a cytokine, an anti-inflammatory molecule, an RNAi, an antibiotic, or an antibody- antibiotic conjugate.
  • the agent is an antibody or fragment thereof, a peptide, or a vaccine.
  • the single domain antibody is genetically fused or chemically conjugated to the agent.
  • a system for providing a molecule to lamina intestinal or gastrointestinal tract of a subject comprising a molecule suitable for administering to the subject, the molecule comprising an agent and a single domain antibody that binds to pIgR, wherein the molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery, or a combination thereof.
  • the agent is a diabetes medication.
  • the diabetes medication is selected from a group consisting of insulin, glucagon-like-peptide-1, insulin-mimic peptides, and glucagon-like-peptide-1-mimic peptides.
  • the agent is a peptide or an antibody or a fragment thereof.
  • the antibody or fragment thereof is selected from a group consisting of an anti-TNF-alpha antibody or a fragment thereof, an anti-IL23 antibody or a fragment thereof, and an antibody that binds to a receptor of IL23 or a fragment thereof.
  • the agent is a vaccine.
  • the vaccine is for preventing an infection selected from a group consisting of Vibrio, Cholera, Typhoid, Rotavirus, Tuberculosis, HIV, Flu, Ebola, and Sendai.
  • a system comprising a means for providing a molecule to lamina limbal or gastrointestinal tract of a subject.
  • the molecule comprises an agent and a single domain antibody that binds to pIgR.
  • the agent is an antibody or fragment thereof, a peptide, a vaccine, a small molecule, a polynucleotide, a radioisotope, a toxin, an enzyme, an anticoagulant, a hormone, a cytokine, an anti-inflammatory molecule, an RNAi, an antibiotic, or an antibody- antibiotic conjugate.
  • the agent is an antibody or fragment thereof, a peptide, or a vaccine.
  • the single domain antibody is genetically fused or chemically conjugated to the agent.
  • the single domain antibody binds to an extracellular domain 1, an extracellular domain 2, an extracellular domain 1-2, an extracellular domain 3, an extracellular domain 2-3, an extracellular domain 4-5, or an extracellular domain 5 of pIgR.
  • the single domain antibody binds to an extracellular domain 1 of pIgR.
  • the single domain antibody binds to an extracellular domain 2 of pIgR.
  • the single domain antibody binds to an extracellular domain 1-2 of pIgR. In some embodiments, the single domain antibody binds to an extracellular domain 3 of pIgR. In some embodiments, the single domain antibody binds to an extracellular domain 2-3 of pIgR. In some embodiments, the single domain antibody binds to an extracellular domain 4-5 of pIgR. In some embodiments, the single domain antibody binds to an extracellular domain 5 of pIgR. [0036] In some embodiments, the single domain antibody competes with IgA binding to the pIgR. In some embodiments, the single domain antibody promotes IgA binding to the pIgR.
  • the KD of the binding of the single domain antibody to pIgR is from about 4 to about 525 nM. In some embodiments, the K D of the binding of the single domain antibody to pIgR is less than about 50 nM. In some embodiments, the KD of the binding of the single domain antibody to pIgR is from about 4 to about 34 nM. [0038] In some embodiments, the T m of the single domain antibody is from about 53 to about 77 °C. In other embodiments, the T m of the single domain antibody is from 53.9 to 76.4 °C. [0039] In some embodiments, pIgR is human pIgR.
  • pIgR is mouse pIgR.
  • the single domain antibody provided herein does not bind to a stalk sequence of human pIgR (e.g., SEQ ID NO:143 and/or a stalk sequence of mouse pIgR (e.g., SEQ ID NO:144 or SEQ ID NO:145).
  • the single domain antibody comprises a CDR3 sequence of GSIDLNWYGGMDY (SEQ ID NO: 60), TTVLTDPRVLNEYAT (SEQ ID NO: 61), DVFGSSGYVETY (SEQ ID NO: 62), PLTAR (SEQ ID NO: 63), DPFNQGY (SEQ ID NO: 64), PLTSR (SEQ ID NO: 65), MVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 66), DQRGY (SEQ ID NO: 67), QRGY (SEQ ID NO: 271), DPFNQGY (SEQ ID NO: 68), DLAEYSGTYSSPADSPAGYDY (SEQ ID NO: 69), ARYYVSGTYFPANY (SEQ ID NO: 70), GSIDLNWYGGMDY (SEQ ID NO: 71), SIDLNWYGGMD (SEQ ID NO: 272), TTVLTDPRVLNEY
  • the single domain antibody comprises a CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 30), RINGGGITHYAESVKG (SEQ ID NO: 31), FIDRIATTTIATSVKG (SEQ ID NO: 32), AITWNGGTTYYADSVKG (SEQ ID NO: 33), FISGGGTTTYADSVKG (SEQ ID NO: 34), RITGGGSTHYAESVKG (SEQ ID NO: 35), AISWSGGSTTYADPVKG (SEQ ID NO: 36), AISWSGSSAGYGDSVKG (SEQ ID NO: 37), AIRWSGGRTLYADSVKG (SEQ ID NO: 38), SITWNGGSTSYADSVKG (SEQ ID NO: 39), DWNGRGTYY (SEQ ID NO: 40), WNGRGTY (SEQ ID NO: 260), NGGGI (SEQ ID NO: 41), GGG (SEQ ID NO: 261), DRIAT (SEQ ID NO: 42
  • the single domain antibody comprises a CDR1 sequence of SYRMG (SEQ ID NO: 1), INVMG (SEQ ID NO: 2), SNAMG (SEQ ID NO: 3), SYAMG (SEQ ID NO: 4), SDAMG (SEQ ID NO: 5), INVMG (SEQ ID NO: 6), TYRMG (SEQ ID NO: 7), RYAMG (SEQ ID NO: 8), FTTYRMG (SEQ ID NO: 258), TYRMG (SEQ ID NO: 259), FNTYAMG (SEQ ID NO: 9), GLTFSSY (SEQ ID NO: 10), GSIFSIN (SEQ ID NO: 11), GTSVSSN (SEQ ID NO: 12), GRTFSSY (SEQ ID NO: 13), GSSVSSD (SEQ ID NO: 14), RSIGSIN (SEQ ID NO: 15), GRTFSTY (SEQ ID NO: 16), GFTFTRY (SEQ ID NO: 17), GRTFTTY (SEQ ID NO: 14),
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: a) VHH1: i) the CDR1 sequence of SYRMG (SEQ ID NO: 1), the CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 30), and the CDR3 sequence of GSIDLNWYGGMDY (SEQ ID NO: 60); ii) the CDR1 sequence of GLTFSSY (SEQ ID NO: 10), the CDR2 sequence of DWNGRGTYY (SEQ ID NO: 40) or WNGRGTY (SEQ ID NO: 260), and the CDR3 sequence of GSIDLNWYGGMDY (SEQ ID NO: 71) or SIDLNWYGGMD (SEQ ID NO: 272); iii) the CDR1 sequence of GLTFSSYR (SEQ ID NO: 20),
  • the single domain antibody comprises a framework derived from the framework of any of the single domain antibodys comprising the sequences of QVQLVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGSIDLNWYGGMD YWGQGTQVTVSS (SEQ ID NO: 93), EVQVVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTVYLQMNSLKPEDTAVYYCAATTVLTDPRVLNEYA TWGQGTQVTVSS (SEQ ID NO: 94), QLQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVARINGGGITHY AESVKGRFTISRDNAKNTVYLQ
  • the single domain antibody comprises a framework comprising sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of QVQLVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGSIDLNWYGGMD YWGQGTQVTVSS (SEQ ID NO: 93), EVQVVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTVYLQMNSLKPEDTAVYYCAATTVLTDPRVLNEYA TWGQGTQVTVSS (SEQ ID NO: 94), QLQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQ
  • the single domain antibody is comprised of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of QVQLVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGSIDLNWYGGMD YWGQGTQVTVSS (SEQ ID NO: 93), EVQVVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTVYLQMNSLKPEDTAVYYCAATTVLTDPRVLNEYA TWGQGTQVTVSS (SEQ ID NO: 94), QLQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQ
  • the single domain antibody is genetically fused or chemically conjugated to the agent.
  • the single domain antibody provided herein further comprises a linker between the single domain antibody and the agent.
  • the linker is a polypeptide.
  • the linker is a flexible linker comprising a sequence selected from the group consisting of EPKTPKPQPQPQLQPQPNPTTESKSPK (SEQ ID NO: 130), (EAAAK)n (SEQ ID NO: 147), (GGGGS)n (SEQ ID NO: 148) and (GGGS)n (SEQ ID NO: 149), wherein n is an integer from 1 to 20.
  • the single domain antibody is chemically-conjugated to the agent. In other embodiments, the single domain antibody is non-covalently bound to the agent. [0051] In some embodiments, the method provided herein does not inhibit pIgR-mediated transcytosis of IgA.
  • the single domain antibody comprises a CDR1 sequence of SNAMG (SEQ ID NO: 3), INVMG (SEQ ID NO: 6), TYRMG (SEQ ID NO: 7), RYAMG (SEQ ID NO: 8), FTTYRMG (SEQ ID NO: 258), TYRMG (SEQ ID NO: 259), FNTYAMG (SEQ ID NO: 9), GTSVSSN (SEQ ID NO: 12), GRTFSSY (SEQ ID NO: 13), RSIGSIN (SEQ ID NO: 15), GRTFSTY (SEQ ID NO: 16), GFTFTRY (SEQ ID NO: 17), GRTFTTY (SEQ ID NO: 18), GRTLSFNTY (SEQ ID NO: 19), GTSVSSNA (SEQ ID NO: 22), RSIGSINV (SEQ ID NO: 25), GRTFSTYR (SEQ ID NO: 26), GFTFTRYA (SEQ ID NO: 27), GRTFTTYR (SEQ ID NO: 3), INVMG (SEQ
  • the single domain antibody comprises a CDR2 sequence of FIDRIATTTIATSVKG (SEQ ID NO: 32), RITGGGSTHYAESVKG (SEQ ID NO: 35), AISWSGGSTTYADPVKG (SEQ ID NO: 36), AISWSGSSAGYGDSVKG (SEQ ID NO: 37), AIRWSGGRTLYADSVKG (SEQ ID NO: 38), SITWNGGSTSYADSVKG (SEQ ID NO: 39), DRIAT (SEQ ID NO: 42), RIA (SEQ ID NO: 262), TGGGS (SEQ ID NO: 45), GGG (SEQ ID NO: 265), SWSGGS (SEQ ID NO: 46), WSGG (SEQ ID NO: 266), SWSGSS (SEQ ID NO: 47), WSGS (SEQ ID NO: 267), RWSGGR (SEQ ID NO: 48), WSGG (SEQ ID NO: 268), TWNGGS (SEQ ID NO: 49), WNGG (
  • the single domain antibody comprises a CDR3 sequence of PLTAR (SEQ ID NO: 63), MVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 66), DQRGY (SEQ ID NO: 67), QRGY (SEQ ID NO: 271), DPFNQGY (SEQ ID NO: 68), DLAEYSGTYSSPADSPAGYDY (SEQ ID NO: 69), ARYYVSGTYFPANY (SEQ ID NO: 70), PLTAR (SEQ ID NO: 74), LTA (SEQ ID NO: 275), MVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 77), VNPIITAWGTIGVREIPDYD (SEQ ID NO: 278), DQRGY (SEQ ID NO: 78), RG (SEQ ID NO: 279), DPFNQGY (SEQ ID NO: 79), PFNQG (SEQ ID NO: 280), DLAEYSGTY
  • Figures 1A and 1B are schematics showing the pathway of pIgR-mediated bidirectional transcytosis.
  • Figure 1A shows that molecules binding to the secretory component (domains 1-5) of the pIgR ectodomain, such as dimeric IgA (natural ligand) or VHH (artificial pIgR ligand), can transcytose the epithelial cell from the basolateral to the apical direction and reach the mucosal lumen from blood.
  • This secretory component-mediated forward transport can be used for delivering molecules to the mucosal lumen from systemic circulation. Described herein are VHH molecules that bind to the secretory component and transcytose from the basolateral to the apical side of the epithelium.
  • Figure 1B shows that molecules binding to the stalk region of the pIgR ectodomain (any artificial ligand) can transcytose the epithelial cell from the apical to the basolateral direction and reach the blood from mucosal lumen.
  • This stalk- mediated reverse transport can be used for delivering molecules to systemic circulation following oral consumption.
  • Figure 2 illustrates data on epitope mapping of pIgR binders.
  • HIS-tagged pIgR constructs (D1, D2, D3, D4, D5, D1-D2, D2-D3, D3-D4 and D4-D5 were expressed and purified from HEK293 cells using immobilized metal ion affinity chromatography. Because the expression and purification yield were very low for two constructs (D4 and D3-D4), these were not used for binding studies.
  • the heat map of Figure 2 shows the binding of VHH-mono-Fc molecules to immobilized pIgR constructs in electrochemiluminescence units. K D values for all positive interactions were measured by bio-layer interferometry.
  • FIG. 2 The heat map of Figure 2 indicates that the epitopes of VHH2 and VHH3 are primarily contained within hpIgR domain-1, the epitopes of VHH4 and VHH6 are primarily contained within hpIgR domain-2, and the epitopes of other six VHHs are primarily contained within hpIgR domains 4-5.
  • Figures 3A-3B illustrate data on the effect of VHH on IgA binding to hpIgR-ECD.
  • Figure 3A shows KD values for full-length hpIgR ECD binding to immobilized VHH-mono-Fc in the absence (blue) and presence (red) of dIgA2.
  • Figure 3B shows the K D values for immobilized dIgA2 binding to hpIgR ectodomain with and without the presence of VHH-mono- Fc molecules.
  • dIgA2 was immobilized using amine-reactive biosensors, and the binding of pIgR and pIgR-VHH complexes were measured by bio-layer interferometry.
  • Three molecules (VHH2, VHH3 and VHH5) had a negative effect on IgA binding to pIgR. Other VHH molecules display a small positive effect on IgA binding to pIgR.
  • Figure 4 depicts the results of assays on the transcytosis activity of VHH-mono-Fc molecules.
  • the top panel is a schematic of the EpiAirway primary human lung tissue model used for assaying VHH transcytosis.
  • the meso-scale discovery (MSD) assay was developed to quantify the amount of VHH present in the basolateral and apical chambers before and after transcytosis.
  • a biotinylated anti-VHH antibody was used to capture VHH-mono-Fc molecules on streptavidin plates and a ruthenylated anti-human-Fc antibody was used as a detection antibody.
  • the bottom panel is a graph showing the amount of VHH present in the apical mucus 24 hours post VHH treatment.
  • Figure 5 illustrates data showing tracking pIgR and VHH across the primary human lung tissue model.
  • the left panel of Figure 5 is a heatmap showing the amount of pIgR retained on the EpiAirway primary human lung tissue model following transcytosis.
  • the right panel of Figure 5 is a heatmap showing the amount of VHH retained on the EpiAirway primary human lung tissue model following transcytosis. Following 48 hours post-treatment, tissue samples were fixed, permeabilized and stained for hpIgR and VHH.
  • FIG. 5 shows that VHHs displayed distinct profiles of pIgR and VHH distribution across the tissue depth dimension.
  • Figure 5 also shows that Among the five VHHs that showed potent transcytosis, VHH2, VHH9 and VHH12-treated tissue models showed higher VHH staining near the apical surface than the other VHHs.
  • VHH6-treated model showed the lowest staining for both VHH and pIgR across the tissue thickness. Imaging studies corroborated transcytosis results and showed colocalization of hpIgR and VHH, especially closer to the apical epithelium.
  • Figure 6A is a schematic showing the structure of pIgR.
  • Figure 6B is a schematic showing a mechanism of pIgR-mediated transport.
  • Figure 7 shows the expression of pIgR in various organs.
  • Figure 8 shows selection criteria used to assess VHH molecules that were generated from mpIgR antigen.
  • Figure 9 shows selection criteria used to assess VHH molecules that were generated from hpIgR antigen.
  • Figure 10 shows the results of an assay for ability of VHH molecules to bind to MDCK cells expressing pIgR.
  • Figure 11 shows the expression of hpIgR on MDCK cells. Staining shows hpIgR located on the surface and interior of the monolayer of MDCK cells. The distribution of hpIgR staining within the monolayer is not uniform. Initial experiments show hpIgR receptor density at about 6000 on the surface per cell. The blue color indicates Hoechst stain for nucleus, the green color indicates antibody staining, and the red indicates anti-Rab5 staining.
  • Figures 12A-12B show the results of a VHH transcytosis assay using MDCK-hpIgR cells, as described in Example 3. Apical VHH amounts at 0, 24, and 48 hours are shown in Figure 12B, left panel.
  • FIG. 12B Fold increase in apical VHH amounts at 24 hours relative to a control VHH is shown in Figure 12B, right panel.
  • Figure 12C shows transcytosis activity of VHH-mono-Fc molecules across MDCK- hpIgR monolayers from the basolateral to the apical chamber. Fold increase in apical VHH amounts at 24 hours relative to control VHH is shown.
  • Figure 13 shows sequence characteristics of a set of VHH molecules, with regions of highly conserved sequence similarity are shown (SEQ ID NOS.: 93-95, 97-103 and 247-249).
  • Figure 14 is a chart summarizing the purification of VHH molecules.
  • Figure 15 shows the results for A-SEC purification of VHH molecules.
  • Figure 16 shows the results for SEC-MALS analysis of VHH molecules.
  • Figure 17 shows the results of a thermal stability assay of VHH molecules by differential scanning fluorimetry (DSF).
  • Figure 18 depicts the EpiAirway human tissue model.
  • Figure 19 shows the results of a VHH transcytosis assay using the EpiAirway model. The left panel shows a heat map of the amount of each tested VHH in the apical mucus at 0, 24 and 48 hours. Electrochemiluminescence (ECLU) unites obtained from the MSD assay was plotted as a heat map.
  • ECLU Electrochemiluminescence
  • the top right panel shows the amount of VHH in the apical mucus at 24 hours, and bottom right panel shows the fold increase of VHH over control in the apical mucus.
  • the top right panel shows that five VHHs (VHH2, VHH6, VHH9, VHH11 and VHH12) showed >20-fold increase in their mucosal amount relative to control VHH molecules, and also that VHH12 showed 38-fold increase in mucus relative to control VHH and displayed the highest transcytosis activity.
  • Figure 20 shows the results of IgA transcytosis assay using the EpiAirway model.
  • Figure 20 shows that VHH2 and VHH12-treated tissue samples stained strongly for VHH and colocalized with pIgR relative to VHH3 and VHH14 (negative control).
  • Figure 21 shows colocalization of hpIgR and VHH.
  • Figure 22 shows 3D reconstruction shows localization of hpIgR and VHH to the apical surface of the EpiAirway model.
  • Figure 23 shows that the EpiAirway tissue model is on a slanted membrane.
  • Figure 24 illustrates a strategy for Opera Phenix imaging and analysis to overcome slanted tissue issues with EpiAirway tissue model.
  • Figure 25 shows the crystal structure of unliganded hpIgR in an inactive conformation.
  • Figure 26 shows structure of pIgR:IgA complex by constrained scattering modeling. The figure is adapted from Bonner et al., J. Biol. Chem., 2009, 284(8):5077-87.
  • Figure 27A shows a structural model for IgA transcytosis. The figure is adapted from Stadtmueller et al., Elife, March 4, 2016, e10640.
  • Figure 27B shows a schematic of pIgR-mediated dimeric IgA transport across the mucosal epithelial barrier.
  • IgA production by plasma cells and IgA dimerization (1) IgA production by plasma cells and IgA dimerization; (2) Binding of dimeric IgA (dIgA) to pIgR ECD on the basolateral side of the epithelium (pIgR-dIgA interactions are mediated by domains 1 and 5 of pIgR and Fc and J chains of dIgA); (3) pIgR- mediated transcytosis of dimeric IgA (clathrin-mediated endocytosis drives the basolateral to apical transport, and upon reaching the apical side, pIgR ECD is proteolytically cleaved and released into mucus along with IgA.
  • FIG. 28A-28D show the effect of IgA on VHH binding to hpIgR.
  • Figure 29 shows the results of domain-level epitope mapping of pIgR binders VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH7, VHH9, VHH10, VHH11 and VHH12.
  • the top panel cartoon is adapted from Stadtmueller et al., Elife, March 4, 2016, e10640.
  • Figure 30A shows binding kinetics for hpIgR D2 binders.
  • Figure 30B shows binding kinetics for hpIgR D4-D5 binders.
  • Figure 31 shows properties of VHH2 and VHH3 (SEQ ID NOS.: 93-95).
  • Figure 32A illustrates structure of domains and sequences of hpIgR and shows that D1 is necessary for IgA binding to hpIgR. The figure is adapted from Stadtmueller et al., Elife, March 4, 2016, e10640 (SEQ ID NOS.: 250-252).
  • FIG 32B shows the structure of secretory IgA1 (sIgA1), the complex between dimeric IgA and secretory component, obtained by constrained modelling of solution scattering and AUC information (created from PDB ID 3CHN). Heavy chain is shown in orange, light chain is shown in green, J chain is shown in pink and secretory component is shown in teal. The figure is adapted from Bonner et al., Mucosal Immunol., 2:74-84 (2009).
  • Figures 33A-33D show the results of VHH/IgA competition studies of Example 6.
  • Figure 33A The crystal structures in Figure 33A is adapted from Stadtmueller et al., Elife, March 4, 2016, e10640 (SEQ ID NOS.: 250 and 253-257).
  • Figure 33B shows a cartoon representation of hpIgR domain-1 created from PDB ID 5D4K. CDR1, CDR2 and CDR3 of hpIgR domain-1 are shown in orange, pink and light red, respectively, wherein hpIgR domain-1 CDRs were swapped with corresponding teleost fish CDRs to test the influence of hpIgR domain-1 CDRs on VHH binding.
  • Figure 33C shows IgA binding to immobilized pIgR constructions, including KD values (KD, Kon, or Koff).
  • Figure 33D shows kinetic parameters for VHH2 and VHH3 binding to sensor immobilized HIS-tagged pIgR protein constructs.
  • the KD, Kon, or Koff are shown in the lower left, upper left and upper right panels, respectively.
  • Figure 33D shows that the hD1_tCDR2 construct did not show binding to both VHH2 and VHH3. Binding kinetic parameters were obtained by bio-layer interferometry, and the fold change in KD values for VHH2 and VHH3 binding to pIgR domain constructs relative to full-length hpIgR ECD is shown in shown in the lower right panel.
  • Figure 34 shows data describing how VHH2 and VHH3 compete with one another for binding to pIgR.
  • Figure 35 illustrates that four molecules (VHH3, VHH4, VHH5 and VHH6) recognize buried epitopes on pIgR.
  • Figures 36A-36B shows that VHH3 recognizes a complex epitope on the hpIgR domain-1 interface.
  • Figures 37A-37B show results of VHH-mono-Fc molecules in forward and reverse transcytosis assays using MDCK-hpIgR monolayers, as described in Example 7. These results demonstrate bidirectional transport.
  • Figure 37A shows the results for the forward transcytosis (basolateral to apical direction), wherein 20 mg of test or control VHH-mono-Fc molecules were added to basolateral chamber and fold increase in apical [VHH] over control is shown at 24 hours (light gray) and 48 hours (dark gray) post treatment.
  • VHH-mono-Fc molecules comprising a VHH2, VHH6, VHH9, VHH11 or VHH12 domain showed >20-fold increase in their apical concentration relative to control VHH-mono-Fc molecules at 48 hours, whereas VHH-mono-Fc molecules comprising a VHH4 domain showed a 15-fold increase in its apical concentration.
  • Figure 37B shows the results of reverse transcytosis (apical to basolateral direction), wherein 20 mg of test or control VHH-mono-Fc molecules were added to apical chamber and fold increase in basolateral [VHH] over control is shown at 24 hours (light gray) and 48 hours (dark gray) post treatment.
  • VHH-mono-Fc molecules comprising a VHH6, VHH11 , or VHH12 domain showed >10-fold increase in their basolateral concentration relative to control VHH-mono-Fc molecules at 48 hours.
  • VHH-mono-Fc molecules comprising a VHH2, VHH4 or VHH9 domain showed >5-fold increase in their basolateral concentration relative to control VHH-mono-Fc molecules at 48 hours.
  • Results for Figures 37A and 37B were obtained from three independent experiments, each containing two technical replicates.
  • Figures 38A-38B show results of VHH-mono-Fc molecules in forward and reverse transcytosis assays using MDCK-hpIgR monolayers, as described in Example 7. These resulted demonstrate bidirectional transport.
  • Figure 38B shows the comparison of forward and reverse transport of VHH-mono-Fc molecules at 48 hours post VHH treatment.
  • Figures 39A-39B show results for forward and reverse transcytosis kinetics of VHH- mono-Fc molecules across MDCK-hpIgR monolayers, as described in Example 7.
  • Figure 39A shows the results of forward transcytosis kinetics (basolateral to apical direction), wherein 20 mg of test or control VHH-mono-Fc molecules were added to the basolateral chamber. The amount of VHH present in the apical chamber (mg) was quantified and shown at different time points (0, 4, 8, 12, 24, 36 and 48 hours) post VHH treatment.
  • VHH-mono-Fc molecules increased over time in the apical chamber.
  • >10% of the basolateral VHH input (2 mg) was transported to the apical chamber (except VHH-mono- Fc molecules comprising a VHH3 or VHH7 domain).
  • Figure 39B shows the results of reverse transcytosis kinetics (apical to basolateral direction), wherein 20 mg of test or control VHH- mono-Fc molecules were added to the apical chamber.
  • the amount of VHH present in the basolateral chamber was quantified and shown at different time points (0, 4, 8, 12, 24, 36 and 48 hours) post VHH treatment.
  • VHH-mono-Fc molecules The concentration of VHH-mono-Fc molecules increased over time in the basolateral chamber. For six VHH-mono-Fc molecules, >10% of the apical VHH input (2 mg) was transported to the basolateral chamber (VHH-mono-Fc molecules comprising a VHH2, VHH4, VHH6, VHH9, VHH11 or VHH12 domain). 5.
  • single domain antibodies e.g., VHH domains
  • pIgR polymeric immunoglobulin receptor
  • pIgR polymeric immunoglobulin receptor
  • antibody immunoglobulin
  • Ig immunoglobulin
  • monoclonal antibodies including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies
  • antibody compositions with polyepitopic or monoepitopic specificity polyclonal or monovalent antibodies
  • multivalent antibodies multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity)
  • An antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse and rabbit, etc.
  • antibody is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed.1995); and Kuby, Immunology (3d ed.1997).
  • the specific molecular antigen can be bound by an antibody provided herein, including a polypeptide or an epitope.
  • Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies including from Camelidae species (e.g., llama or alpaca) or their humanized variants, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • Non-limiting examples of functional fragments include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody.
  • scFv single-chain Fvs
  • Fab fragments F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments
  • dsFv disulfide-linked Fvs
  • antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody).
  • an antigen e.g., one or more CDRs of an antibody.
  • antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Plückthun and Skerra, 1989, Meth. Enzymol.178:497-515; and Day, Advanced Immunochemistry (2d ed.1990).
  • the antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.
  • Antibodies may be agonistic antibodies or antagonistic antibodies.
  • Antibodies may be neither agonistic nor antagonistic.
  • An “antigen” is a structure to which an antibody can selectively bind.
  • a target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide.
  • an antigen is associated with a cell, for example, is present on or in a cell.
  • An “intact” antibody is one comprising an antigen-binding site as well as a CL and at least heavy chain constant regions, CH1, CH2 and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof. In certain embodiments, an intact antibody has one or more effector functions.
  • the terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms refer to that portion of a binding molecule, which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g., the CDRs).
  • Antigen-binding fragment as used herein include “antibody fragment,” which comprise a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody.
  • antibody fragments include, without limitation, Fab, Fab’, F(ab’)2, and Fv fragments; diabodies and di-diabodies (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci.90:6444-48; Lu et al., 2005, J. Biol. Chem.280:19665-72; Hudson et al., 2003, Nat. Med.9:129-34; WO 93/11161; and U.S. Pat.
  • Single domain antibody refers to a single monomeric variable antibody domain and which is capable of antigen binding (e.g., single domain antibodies that bind to pIgR).
  • Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine.
  • a single domain antibody can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco, as described herein.
  • Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; VHHs derived from such other species are within the scope of the disclosure.
  • the single domain antibody e.g., VHH
  • the single domain antibody has a structure of FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4.
  • Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein.
  • binding refers to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope.
  • the ratio of dissociation rate (koff) to association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen (koff/kon) is the dissociation constant KD, which is inversely related to affinity.
  • K D the dissociation constant
  • the value of K D varies for different complexes of antibody and antigen and depends on both k on and k off .
  • the dissociation constant KD for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art.
  • the affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen.
  • binding molecules described herein terms such as “bind to,” “that specifically bind to,” and analogous terms are also used interchangeably herein and refer to binding molecules of antigen binding domains that specifically bind to an antigen, such as a polypeptide.
  • a binding molecule or antigen binding domain that binds to or specifically binds to an antigen may be cross-reactive with related antigens.
  • a binding molecule or antigen binding domain that binds to or specifically binds to an antigen does not cross-react with other antigens.
  • a binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, Octet ® , Biacore ® , or other techniques known to those of skill in the art.
  • a binding molecule or antigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISAs).
  • RIA radioimmunoassays
  • ELISAs enzyme linked immunosorbent assays
  • a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed.1989) for a discussion regarding binding specificity.
  • the extent of binding of a binding molecule or antigen binding domain to a “non- target” protein is less than about 10% of the binding of the binding molecule or antigen binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA.
  • FACS fluorescence activated cell sorting
  • RIA fluorescence activated cell sorting
  • Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • a binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the binding molecule is useful, for example, as a diagnostic agent in targeting the antigen.
  • a binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (K D ) of less than or equal to 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM.
  • K D dissociation constant
  • a binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species (e.g., between human and cyno species).
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (KD).
  • KD dissociation constant
  • Affinity can be measured by common methods known in the art, including those described herein. Low- affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high- affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following.
  • the “KD” or “KD value” may be measured by assays known in the art, for example by a binding assay.
  • the KD may be measured in a RIA, for example, performed with the Fab version of an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865- 81).
  • the KD or KD value may also be measured by using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, an Octet®Red96 system, or by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000.
  • BLI biolayer interferometry
  • SPR surface plasmon resonance
  • an “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the Octet®Red96, the Biacore®TM-2000, or the Biacore®TM-3000 system.
  • BLI biolayer interferometry
  • SPR surface plasmon resonance
  • the binding molecules or antigen binding domains can comprise “chimeric” sequences in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No.4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences may include humanized sequences.
  • the binding molecules or antigen binding domains can comprise portions of “humanized” forms of nonhuman (e.g., camelid, murine, non-human primate) antibodies that include sequences from human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as camelid, mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • a nonhuman species e.g., donor antibody
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody.
  • a humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the binding molecules or antigen binding domains can comprise portions of a “fully human antibody” or “human antibody,” wherein the terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region.
  • the binding molecules may comprise a single domain antibody sequence.
  • the terms refer to an antibody that comprises a variable region and constant region of human origin.
  • Fully human antibodies in certain embodiments, can also encompass antibodies which bind polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence.
  • the term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242).
  • a “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies.
  • Human antibodies specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol.227:381; Marks et al., 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1: 755-68). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., 1991, J. Immunol.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol.6(5):561- 66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol.8(4):455-58; and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE TM technology).
  • the binding molecules or antigen binding domains can comprise portions of a “recombinant human antibody,” wherein the phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see e.g., Taylor, L.
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the binding molecules or antigen binding domains can comprise a portion of a “monoclonal antibody,” wherein the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts or well-known post-translational modifications such as amino acid iomerizatio or deamidation, methionine oxidation or asparagine or glutamine deamidation, each monoclonal antibody will typically recognize a single epitope on the antigen.
  • a “monoclonal antibody,” as used herein is an antibody produced by a single hybridoma or other cell.
  • the term “monoclonal” is not limited to any particular method for making the antibody.
  • the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., 1975, Nature 256:495, or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No.4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol. 222:581-97, for example.
  • a typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and g chains and four CH domains for m and e isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
  • the VL is aligned with the VH
  • the CL is aligned with the first constant domain of the heavy chain (CH1).
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • the pairing of a VH and VL together forms a single antigen-binding site.
  • Fab refers to an antibody region that binds to antigens.
  • a conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain.
  • variable region and the constant region of the heavy chain in a Fab region are VH and CH1 regions
  • variable region and the constant region of the light chain in a Fab region are VL and CL regions.
  • the VH, CH1, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability according to the present disclosure.
  • VH and CH1 regions can be on one polypeptide
  • VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG.
  • VH, CH1, VL and CL regions can all be on the same polypeptide and oriented in different orders as described in more detail the sections below.
  • variable region refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variable region of the heavy chain may be referred to as “VH.”
  • the variable region of the light chain may be referred to as “VL.”
  • variable refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variable regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions that are each about 9-12 amino acids long.
  • the variable regions of heavy and light chains each comprise four FRs, largely adopting a b sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the b sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed.1991)).
  • the constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
  • the variable regions differ extensively in sequence between different antibodies.
  • the variable region is a human variable region.
  • variable region residue numbering refers to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra).
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra).
  • the “EU index as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody.
  • the term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (d), epsilon (e), gamma (g), and mu ( ⁇ ), based on the amino acid sequence of the heavy chain constant region.
  • the distinct heavy chains differ in size: a, d, and g contain approximately 450 amino acids, while ⁇ and e contain approximately 550 amino acids.
  • these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.
  • the term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • kappa kappa
  • l lambda
  • the terms “hypervariable region,” “HVR,” “Complementarity Determining Region,” and “CDR” are used interchangeably.
  • CDR refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH b-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL b-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. [00123] CDR regions are well known to those skilled in the art and have been defined by well- known numbering systems. For example, the Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra).
  • Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol.196:901-17).
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol.2 (Kontermann and Dübel eds., 2d ed.2010)).
  • the “contact” hypervariable regions are based on an analysis of the available complex crystal structures.
  • Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information System ® (Lafranc et al., 2003, Dev. Comp. Immunol.27(1):55-77).
  • IMGT is an integrated information system specializing in immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complex (MHC) of human and other vertebrates.
  • CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain.
  • location of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody.
  • CDR complementary determining region
  • individual CDRs e.g., “CDR-H1, CDR-H2
  • the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR is given.
  • Hypervariable regions may comprise “extended hypervariable regions” as follows: 24- 36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.
  • the term “constant region” or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • variable region refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site.
  • the constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.
  • framework or “FR” refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies (e.g., single domain antibodies), diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl- terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region.
  • exemplary “effector functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor), etc.
  • effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those skilled in the art.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide.
  • the variant Fc region herein can possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.
  • an “epitope” is a term in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind.
  • An epitope can be a linear epitope or a conformational, non- linear, or discontinuous epitope.
  • an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope).
  • a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure.
  • a binding molecule binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure.
  • a binding molecule requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.
  • alter or “promote,” or “increase” or “expand” or “improve” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule/composition.
  • a measurable physiological response may include but is not limited to an increase in forward or reverse transcytosis, among others apparent from the understanding in the art and the description herein.
  • an “increased” or “enhanced” amount can be a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) the response produced by vehicle or a control composition.
  • times e.g., 500, 1000 times
  • the terms “polypeptide” and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a “polypeptide” can occur as a single chain or as two or more associated chains.
  • vector refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, in order to introduce a nucleic acid sequence into a host cell.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selection control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • the term “host” as used herein refers to an animal, such as a mammal (e.g., a human).
  • the term “host cell” as used herein refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • one or more nucleic acid molecules encoding a single domain antibody or an antibody as described herein are isolated or purified.
  • nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure molecule may include isolated forms of the molecule.
  • Polynucleotide or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • Oligonucleotide refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length.
  • oligonucleotide and “polynucleotide” are not mutually exclusive.
  • a cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced.
  • the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • RNA transcripts The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.”
  • upstream sequences sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.”
  • an operatively linked promoter, enhancer elements, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA).
  • operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame).
  • an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.
  • compositions, or vehicles such as a liquid or solid filler, diluent, solvent, or encapsulating material.
  • Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof.
  • the term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.
  • excipients are pharmaceutically acceptable excipients.
  • Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • a pharmaceutically acceptable excipient is an aqueous pH buffered solution.
  • excipients are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions.
  • An excipient can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • compositions can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • Compositions, including pharmaceutical compounds may contain a binding molecule (e.g., an antibody), for example, in isolated or purified form, together with a suitable amount of excipients.
  • a binding molecule e.g., an antibody
  • the term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a single domain antibody or a therapeutic molecule comprising an agent and the single domain antibody or pharmaceutical composition provided herein which is sufficient to result in the desired outcome.
  • the terms “subject” and “patient” may be used interchangeably.
  • a subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human).
  • the subject is a human.
  • the subject is a mammal, e.g., a human, diagnosed with a condition or disorder.
  • the subject is a mammal, e.g., a human, at risk of developing a condition or disorder.
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.
  • treat refers to the reduction or amelioration of the progression, severity, and/or duration of a disease or condition resulting from the administration of one or more therapies.
  • Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder.
  • Treating includes both managing and ameliorating the disease.
  • the terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy which does not necessarily result in a cure of the disease.
  • the terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., diabetes or a cancer).
  • single Domain Antibodies 5.2.1 Single Domain Antibodies Targeting pIgR [00154] Provided herein are single domain antibodies (e.g., VHH domains) capable of binding to polymeric immunoglobulin receptor (pIgR), that can act as a delivery domain for therapeutic agents.
  • single domain antibodies e.g., VHH domains
  • pIgR polymeric immunoglobulin receptor
  • the single domain antibodies (e.g., VHH domains) provided herein bind to human pIgR (Genbank ID: CR749533) (see Turula, H. & Wobus, C.E. The Role of the Polymeric Immunoglobulin Receptor and Secretory Immunoglobulins during Mucosal Infection and Immunity. Viruses 10 (2018)).
  • the single domain antibodies (e.g., VHH domains) provided herein bind to mouse pIgR.
  • Human pIgR (hpIgR) is an 82 kDa, single-pass transmembrane receptor containing a 620-residue extracellular domain (ECD), a 23-residue transmembrane domain and a 103-residue intracellular domain.
  • ECD extracellular domain
  • pIgR transports soluble polymeric forms of IgA and IgM into apical mucosal tissues from the basolateral side of the epithelium. The process of transporting polymeric immunoglobulins from the basolateral to apical side is transcytosis.
  • pIgR ECD that contains five domains (secretory component) is proteolytically cleaved and released into mucus with or without IgA.
  • pIgR has several different functions that include, but are not limited to, conferring stability to IgA, immune exclusion, anti- inflammatory properties and homeostasis of commensals in the mucosal immune system.
  • Approximately 75% of total daily antibody production is directed to IgA molecules.
  • IgA1 and IgA2 IgA2m(1) and (2) allotypes.
  • IgA1 has elongated hinge region lacking in IgA2, that contains several O-glycan sites and is susceptible to proteolytic cleavage. Endogenous IgA is present in various forms in a compartment-dependent manner.
  • Monomeric IgA (mIgA) is the predominant form in serum (at a concentration of 1-3 mg/mL), primarily as IgA1 (about 90%) produced in bone marrow.
  • Dimeric IgA (dIgA) is formed via S-S bridging of the C-terminal Fc tailpiece with J chain. dIgA is produced locally at target site of action and transported across mucosal surface into secretions of respiratory, GI and genitourinary tracts.
  • Secretory IgA is formed via dIgA complex with extracellular domain of polymeric Ig receptor (pIgR). Cleavage of secretory component (SC) at the mucosal surface of epithelial cells releases S-IgA.
  • the polymeric immunoglobulin receptor (pIgR) binds to soluble dimeric IgA via Fc and J-chain mediated interactions. pIgR does not bind or transport IgG molecules across mucosal epithelium. Though IgG molecules lack a lumen-targeted active transport mechanism, conferring pIgR-binding abilities to IgG can mediate selective transport of IgG antibodies into the mucosal lumen.
  • FIG. 6A The structure of pIgR is summarized in Figure 6A.
  • FIG. 6B A mechanism of pIgR-mediated transport is summarized in Figure 6B.
  • the expression of pIgR in various organs is shown in Figure 7.
  • the single domain antibodies provided herein transport from an apical surface to a basolateral surface (reverse transcytosis) as well as from the basolateral to apical side (transcytosis).
  • the single domain antibody e.g., VHH domain
  • the single domain antibody provided herein competes with IgA binding to the pIgR.
  • the single domain antibody (e.g., VHH domain) provided herein promotes IgA binding to the pIgR.
  • the KD of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is from 4 to 525 nM. In some embodiments, the K D of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is less than 525 nM. In some embodiments, the KD of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is less than 400 nM.
  • the K D of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is less than 350 nM. In some embodiments, the KD of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is less than 300 nM. In some embodiments, the K D of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is less than 250 nM. In some embodiments, the K D of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is less than 200 nM.
  • the K D of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is less than 150 nM. In some embodiments, the K D of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is less than 100 nM. In some embodiments, the KD of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is less than 50 nM. In some embodiments, the K D of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is from 4 to 525 nm.
  • the KD of the binding of the single domain antibody (e.g., VHH domain) provided herein to pIgR is from 4 to 34 nm. Bio-layer interferometry experiments described herein show 8 VHH domain binders having KD values of ⁇ 50 nM for binding to the human pIgR ectodomain (see Table 1). [00163]
  • the T m of the single domain antibody (e.g., VHH domain) is from 53 to 77 °C. In some embodiments, the T m of the single domain antibody (e.g., VHH domain) is from 53.9 to 76.4 °C.
  • the T m of the single domain antibody (e.g., VHH domain) is from 61 to 77 °C. In some embodiments, the T m of the single domain antibody (e.g., VHH domain) is from 61 to 71 °C.
  • the EC50 value for single domain antibody (e.g., VHH domain) binding to an MDCK-hpIgR cell is less than 10 nM. Six such binders comprising a VHH domain are described in Table 1.
  • VHH domain that binds to domain 1 of pIgR, wherein the VHH domain comprises the CDR1, CDR2 and/or CDR3 sequence of VHH2 or VHH3 described herein.
  • the VHH domain that bind to domain 1 of pIgR comprises the CDR1, CDR2 and CDR3 sequence of: VHH2: i) the CDR1 sequence of SYRMG (SEQ ID NO: 1), the CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 30), and the CDR3 sequence of TTVLTDPRVLNEYAT (SEQ ID NO: 61); ii) the CDR1 sequence of GLTFSSY (SEQ ID NO: 10), the CDR2 sequence of DWNGRGTYY (SEQ ID NO: 40) or WNGRGTY (SEQ ID NO: 260), and the CDR3 sequence of TTVLTDPRVLNEYAT (SEQ ID NO: 72) or TVLTDPRVLNEYA (SEQ ID NO: 273); iii) the CDR1 sequence of GLTFSSYR (SEQ ID NO: 20), the CDR2 sequence of IDWNGRGTYY (SEQ ID NO:
  • VHH domain that binds to domain 2 of pIgR, wherein the VHH domain comprises the CDR1, CDR2 and/or CDR3 sequence of VHH4 or VHH6 described herein.
  • the VHH domain that bind to domain 2 of pIgR comprises the CDR1, CDR2 and CDR3 sequence of: VHH4: i) the CDR1 sequence of SNAMG (SEQ ID NO: 3), the CDR2 sequence of FIDRIATTTIATSVKG (SEQ ID NO: 32), and the CDR3 sequence of PLTAR (SEQ ID NO: 63); ii) the CDR1 sequence of GTSVSSN (SEQ ID NO: 12), the CDR2 sequence of DRIAT (SEQ ID NO: 42) or RIA (SEQ ID NO: 262), and the CDR3 sequence of PLTAR (SEQ ID NO: 74) or LTA (SEQ ID NO: 275); iii) the CDR1 sequence of GTSVSSNA (SEQ ID NO: 22), the CDR2 sequence of IDRIATT (SEQ ID NO: 52), and the CDR3 sequence of NHPLTAR (SEQ ID NO: 85); iv) the CDR1 sequence of
  • VHH domain that binds to domain 4-5 of pIgR, wherein the VHH domain comprises the CDR1, CDR2 and/or CDR3 sequence of VHH5, VHH7, VHH9, VHH10 or VHH11 described herein.
  • the VHH domain that bind to domain 4-5 of pIgR comprises the CDR1, CDR2 and CDR3 sequence of: VHH5: i) the CDR1 sequence of SYAMG (SEQ ID NO: 4), the CDR2 sequence of AITWNGGTTYYADSVKG (SEQ ID NO: 33), and the CDR3 sequence of DPFNQGY (SEQ ID NO: 64); ii) the CDR1 sequence of GRTFSSY (SEQ ID NO: 13), the CDR2 sequence of TWNGGT (SEQ ID NO: 43) or WNGG (SEQ ID NO: 263), and the CDR3 sequence of DPFNQGY (SEQ ID NO: 75) or PFNQG (SEQ ID NO: 276); iii) the CDR1 sequence of GRTFSSYA (SEQ ID NO: 23), the CDR2 sequence of ITWNGGTT (SEQ ID NO: 53), and the CDR3 sequence of AADPFNQGY (
  • VHH domain that binds to domain 5 of pIgR comprises the CDR1, CDR2 and/or CDR3 sequence of VHH12 described herein.
  • the VHH domain that bind to domain 5 of pIgR comprises the CDR1, CDR2 and CDR3 sequence of: VHH12: i) the CDR1 sequence of FNTYAMG (SEQ ID NO: 9), the CDR2 sequence of SITWNGGSTSYADSVKG (SEQ ID NO: 39), and the CDR3 sequence of ARYYVSGTYFPANY (SEQ ID NO: 70); ii) the CDR1 sequence of GRTLSFNTY (SEQ ID NO: 19), the CDR2 sequence of TWNGGS (SEQ ID NO: 49) or WNGG (SEQ ID NO: 269), and the CDR3 sequence of ARYYVSGTYFPANY (SEQ ID NO: 81) or RYY
  • the single domain antibodies provide herein are VHH domains.
  • VHH domains are generated as described below, and these VHH domains (referred to as mpIgR_011, hpIgR_021, hpIgR_073, hpIgR_175, hpIgR_181, hpIgR_198, hpIgR_201, hpIgR_221, hpIgR_225, hpIgR_250, hpIgR_266, mpIgR_338, and hpIgR_349) share some sequence characteristics, as shown in Figure 13. Regions of highly conserved sequence similarity are shown in yellow.
  • mpIgR_011 is VHH1
  • hpIgR_021 is VHH3
  • hpIgR_073 is VHH4
  • hpIgR_175 is VHH5
  • hpIgR_181 is VHH6
  • hpIgR_198 is VHH7
  • hpIgR_201 is VHH8
  • hpIgR_221 is VHH9
  • hpIgR_225 is VHH10
  • hpIgR_250 is VHH11
  • hpIgR_266 is VHH12
  • mpIgR_338 is VHH2.
  • the single domain antibody provided herein comprises one or more CDR sequences of any one of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH7, VHH9, VHH10, VHH11, and VHH12.
  • a single domain antibody that binds to pIgR comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the CDR sequences are selected for those in VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH7, VHH9, VHH10, VHH11, and VHH12.
  • CDR1 has an amino acid sequence selected from a group consisting of SYRMG (SEQ ID NO: 1), INVMG (SEQ ID NO: 2), SNAMG (SEQ ID NO: 3), SYAMG (SEQ ID NO: 4), SDAMG (SEQ ID NO: 5), INVMG (SEQ ID NO: 6), TYRMG (SEQ ID NO: 7), RYAMG (SEQ ID NO: 8), FTTYRMG (SEQ ID NO: 258), TYRMG (SEQ ID NO: 259), FNTYAMG (SEQ ID NO: 9), GLTFSSY (SEQ ID NO: 10), GSIFSIN (SEQ ID NO: 11), GTSVSSN (SEQ ID NO: 12), GRTFSSY (SEQ ID NO: 13),
  • a single domain antibody that binds to pIgR comprising a variable region (e.g.,VH) comprising CDR1, CDR2, and CDR3 of any one of antibodies as set forth in Table 1.
  • the single domain antibody comprises a CDR1 sequence present in VHH1, e.g., the CDR1 sequence of SYRMG (SEQ ID NO: 1).
  • the single domain antibody comprises a CDR1 sequence present in VHH2, e.g., the CDR1 sequence of SYRMG (SEQ ID NO: 1).
  • the single domain antibody comprises a CDR1 sequence present in VHH3, e.g., the CDR1 sequence of INVMG (SEQ ID NO: 2). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH4, e.g., the CDR1 sequence of SNAMG (SEQ ID NO: 3). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH5, e.g., the CDR1 sequence of SYAMG (SEQ ID NO: 4).
  • the single domain antibody comprises a CDR1 sequence present in VHH6, e.g., the CDR1 sequence of SDAMG (SEQ ID NO: 5). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH7, e.g., the CDR1 sequence of INVMG (SEQ ID NO: 6). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH9, e.g., the CDR1 sequence of TYRMG (SEQ ID NO: 7).
  • the single domain antibody comprises a CDR1 sequence present in VHH10, e.g., the CDR1 sequence of RYAMG (SEQ ID NO: 8). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH11, e.g., the CDR1 sequence of TYRMG (SEQ ID NO: 259). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH12, e.g., the CDR1 sequence of FNTYAMG (SEQ ID NO: 9).
  • the single domain antibody comprises a CDR1 sequence present in VHH1, e.g., the CDR1 sequence of GLTFSSY (SEQ ID NO: 10). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH2, e.g., the CDR1 sequence of GLTFSSY (SEQ ID NO: 10). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH3, e.g., the CDR1 sequence of GSIFSIN (SEQ ID NO: 11).
  • the single domain antibody comprises a CDR1 sequence present in VHH4, e.g., the CDR1 sequence of GTSVSSN (SEQ ID NO: 12). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH5, e.g., the CDR1 sequence of GRTFSSY (SEQ ID NO: 13). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH6, e.g., the CDR1 sequence of GSSVSSD (SEQ ID NO: 14).
  • the single domain antibody comprises a CDR1 sequence present in VHH7, e.g., the CDR1 sequence of RSIGSIN (SEQ ID NO: 15). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH9, e.g., the CDR1 sequence of GRTFSTY (SEQ ID NO: 16). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH10, e.g., the CDR1 sequence of GFTFTRY (SEQ ID NO: 17).
  • the single domain antibody comprises a CDR1 sequence present in VHH11, e.g., the CDR1 sequence of GRTFTTY (SEQ ID NO: 18). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH12, e.g., the CDR1 sequence of GRTLSFNTY (SEQ ID NO: 19). [00176] In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH1, e.g., the CDR1 sequence of GLTFSSYR (SEQ ID NO: 20).
  • the single domain antibody comprises a CDR1 sequence present in VHH2, e.g., the CDR1 sequence of GLTFSSYR (SEQ ID NO: 20). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH3, e.g., the CDR1 sequence of GSIFSINV (SEQ ID NO: 21). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH4, e.g., the CDR1 sequence of GTSVSSNA (SEQ ID NO: 22).
  • the single domain antibody comprises a CDR1 sequence present in VHH5, e.g., the CDR1 sequence of GRTFSSYA (SEQ ID NO: 23). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH6, e.g., the CDR1 sequence of GSSVSSDA (SEQ ID NO: 24). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH7, e.g., the CDR1 sequence of RSIGSINV (SEQ ID NO: 25).
  • the single domain antibody comprises a CDR1 sequence present in VHH9, e.g., the CDR1 sequence of GRTFSTYR (SEQ ID NO: 26). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH10, e.g., the CDR1 sequence of GFTFTRYA (SEQ ID NO: 27). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH11, e.g., the CDR1 sequence of GRTFTTYR (SEQ ID NO: 28).
  • the single domain antibody comprises a CDR1 sequence present in VHH12, e.g., the CDR1 sequence of GRTLSFNTYA (SEQ ID NO: 29). [00177] In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH1, e.g., the CDR1 sequence of GLTFSSYRMG (SEQ ID NO: 154). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH2, e.g., the CDR1 sequence of GLTFSSYRMG (SEQ ID NO: 154).
  • the single domain antibody comprises a CDR1 sequence present in VHH3, e.g., the CDR1 sequence of GSIFSINVMG (SEQ ID NO: 155). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH4, e.g., the CDR1 sequence of GTSVSSNAMG (SEQ ID NO: 156). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH5, e.g., the CDR1 sequence of GRTFSSYAMG (SEQ ID NO: 157).
  • the single domain antibody comprises a CDR1 sequence present in VHH6, e.g., the CDR1 sequence of GSSVSSDAMG (SEQ ID NO: 158). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH7, e.g., the CDR1 sequence of RSIGSINVMG (SEQ ID NO: 159). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH9, e.g., the CDR1 sequence of GRTFSTYRMG (SEQ ID NO: 160).
  • the single domain antibody comprises a CDR1 sequence present in VHH10, e.g., the CDR1 sequence of RYAMG GFTFTRYAMG (SEQ ID NO: 161).
  • the single domain antibody comprises a CDR1 sequence present in VHH11, e.g., the CDR1 sequence of GRTFTTYRMG (SEQ ID NO: 162).
  • the single domain antibody comprises a CDR1 sequence present in VHH12, e.g., the CDR1 sequence of GRTLSFNTYAMG (SEQ ID NO: 163).
  • the single domain antibody comprises a CDR1 sequence present in VHH1, e.g., the CDR1 sequence of GLTFSSY SSYRMG (SEQ ID NO: 164). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH2, e.g., the CDR1 sequence of SSYRMG (SEQ ID NO: 164). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH3, e.g., the CDR1 sequence of SINVMG (SEQ ID NO: 165).
  • the single domain antibody comprises a CDR1 sequence present in VHH4, e.g., the CDR1 sequence of SSNAMG (SEQ ID NO: 166). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH5, e.g., the CDR1 sequence of SSYAMG (SEQ ID NO: 167). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH6, e.g., the CDR1 sequence of SSDAMG (SEQ ID NO: 168).
  • the single domain antibody comprises a CDR1 sequence present in VHH7, e.g., the CDR1 sequence of SINVMG (SEQ ID NO: 169). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH9, e.g., the CDR1 sequence of STYRMG (SEQ ID NO: 170). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH10, e.g., the CDR1 sequence of TRYAMG (SEQ ID NO: 171).
  • the single domain antibody comprises a CDR1 sequence present in VHH11, e.g., the CDR1 sequence of TTYRMG (SEQ ID NO: 172).
  • the single domain antibody comprises a CDR1 sequence present in VHH12, e.g., the CDR1 sequence of SFNTYAMG (SEQ ID NO: 173).
  • the single domain antibody comprises a CDR1 sequence present in VHH1, e.g., the CDR1 sequence of GLTFSSYRMG (SEQ ID NO: 174).
  • the single domain antibody comprises a CDR1 sequence present in VHH2, e.g., the CDR1 sequence of GLTFSSYRMG (SEQ ID NO: 174). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH3, e.g., the CDR1 sequence of GSIFSINVMG (SEQ ID NO: 175). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH4, e.g., the CDR1 sequence of GTSVSSNAMG (SEQ ID NO: 176).
  • the single domain antibody comprises a CDR1 sequence present in VHH5, e.g., the CDR1 sequence of GRTFSSYAMG (SEQ ID NO: 177). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH6, e.g., the CDR1 sequence of GSSVSSDAMG (SEQ ID NO: 178). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH7, e.g., the CDR1 sequence of RSIGSINVMG (SEQ ID NO: 179).
  • the single domain antibody comprises a CDR1 sequence present in VHH9, e.g., the CDR1 sequence of GRTFSTYRMG (SEQ ID NO: 180). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH10, e.g., the CDR1 sequence of GFTFTRYAMG (SEQ ID NO: 181). In various embodiments of the aspects described herein the single domain antibody comprises a CDR1 sequence present in VHH11, e.g., the CDR1 sequence of GRTFTTYRMG (SEQ ID NO: 182).
  • the single domain antibody comprises a CDR1 sequence present in VHH12, e.g., the CDR1 sequence of GRTLSFNTYAMG (SEQ ID NO: 183).
  • the single domain antibody comprises a CDR2 sequence present in VHH1, e.g., the CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 30).
  • the single domain antibody comprises a CDR2 sequence present in VHH2, e.g., the CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 30).
  • the single domain antibody comprises a CDR2 sequence present in VHH3, e.g., the CDR2 sequence of RINGGGITHYAESVKG (SEQ ID NO: 31). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH4, e.g., the CDR2 sequence of FIDRIATTTIATSVKG (SEQ ID NO: 32). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH5, e.g., the CDR2 sequence of AITWNGGTTYYADSVKG (SEQ ID NO: 33).
  • the single domain antibody comprises a CDR2 sequence present in VHH6, e.g., the CDR2 sequence of FISGGGTTTYADSVKG (SEQ ID NO: 34).
  • the single domain antibody comprises a CDR2 sequence present in VHH7, e.g., the CDR2 sequence of RITGGGSTHYAESVKG (SEQ ID NO: 35).
  • the single domain antibody comprises a CDR2 sequence present in VHH9, e.g., the CDR2 sequence of AISWSGGSTTYADPVKG (SEQ ID NO: 36).
  • the single domain antibody comprises a CDR2 sequence present in VHH10, e.g., the CDR2 sequence of AISWSGSSAGYGDSVKG (SEQ ID NO: 37). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH11, e.g., the CDR2 sequence of AIRWSGGRTLYADSVKG (SEQ ID NO: 38). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH12, e.g., the CDR2 sequence of SITWNGGSTSYADSVKG (SEQ ID NO: 39).
  • the single domain antibody comprises a CDR2 sequence present in VHH1, e.g., the CDR2 sequence of DWNGRGTYY (SEQ ID NO: 40) or WNGRGTY (SEQ ID NO: 260).
  • the single domain antibody comprises a CDR2 sequence present in VHH2, e.g., the CDR2 sequence of DWNGRGTYY (SEQ ID NO: 40) or WNGRGTY (SEQ ID NO: 260).
  • the single domain antibody comprises a CDR2 sequence present in VHH3, e.g., the CDR2 sequence of NGGGI (SEQ ID NO: 41) or GGG (SEQ ID NO: 261).
  • the single domain antibody comprises a CDR2 sequence present in VHH4, e.g., the CDR2 sequence of DRIAT (SEQ ID NO: 42) or RIA (SEQ ID NO: 262).
  • the single domain antibody comprises a CDR2 sequence present in VHH5, e.g., the CDR2 sequence of TWNGGT (SEQ ID NO: 43) or WNGG (SEQ ID NO: 263).
  • the single domain antibody comprises a CDR2 sequence present in VHH6, e.g., the CDR2 sequence of SGGGT (SEQ ID NO: 44) or GGG (SEQ ID NO: 264).
  • the single domain antibody comprises a CDR2 sequence present in VHH7, e.g., the CDR2 sequence of TGGGS (SEQ ID NO: 45) or GGG (SEQ ID NO: 265).
  • the single domain antibody comprises a CDR2 sequence present in VHH9, e.g., the CDR2 sequence of SWSGGS (SEQ ID NO: 46) or WSGG (SEQ ID NO: 266).
  • the single domain antibody comprises a CDR2 sequence present in VHH10, e.g., the CDR2 sequence of SWSGGS (SEQ ID NO: 47) or WSGS (SEQ ID NO: 267).
  • the single domain antibody comprises a CDR2 sequence present in VHH11, e.g., the CDR2 sequence of RWSGGR (SEQ ID NO: 48) or WSGG (SEQ ID NO: 268).
  • the single domain antibody comprises a CDR2 sequence present in VHH12, e.g., the CDR2 sequence of TWNGGS (SEQ ID NO: 49) or WNGG (SEQ ID NO: 269).
  • the single domain antibody comprises a CDR2 sequence present in VHH1, e.g., the CDR2 sequence of IDWNGRGTYY (SEQ ID NO: 50) or IDWNGRGTYYR (SEQ ID NO: 270).
  • the single domain antibody comprises a CDR2 sequence present in VHH2, e.g., the CDR2 sequence of IDWNGRGTYY (SEQ ID NO: 50) or IDWNGRGTYYR (SEQ ID NO: 270).
  • the single domain antibody comprises a CDR2 sequence present in VHH3, e.g., the CDR2 sequence of INGGGIT (SEQ ID NO: 51).
  • the single domain antibody comprises a CDR2 sequence present in VHH4, e.g., the CDR2 sequence of IDRIATT (SEQ ID NO: 52). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH5, e.g., the CDR2 sequence of ITWNGGTT (SEQ ID NO: 53). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH6, e.g., the CDR2 sequence of ISGGGTT (SEQ ID NO: 54).
  • the single domain antibody comprises a CDR2 sequence present in VHH7, e.g., the CDR2 sequence of ITGGGST (SEQ ID NO: 55). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH9, e.g., the CDR2 sequence of ISWSGGST (SEQ ID NO: 56). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH10, e.g., the CDR2 sequence of ISWSGSSA (SEQ ID NO: 57).
  • the single domain antibody comprises a CDR2 sequence present in VHH11, e.g., the CDR2 sequence of IRWSGGRT (SEQ ID NO: 58). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH12, e.g., the CDR2 sequence of ITWNGGST (SEQ ID NO: 59). [00183] In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH1, e.g., the CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 184).
  • the single domain antibody comprises a CDR2 sequence present in VHH2, e.g., the CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 184). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH3, e.g., the CDR2 sequence of RINGGGITHYAESVKG (SEQ ID NO: 185). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH4, e.g., the CDR2 sequence of FIDRIATTTIATSVKG (SEQ ID NO: 186).
  • the single domain antibody comprises a CDR2 sequence present in VHH5, e.g., the CDR2 sequence of AITWNGGTTYYADSVKG (SEQ ID NO: 187). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH6, e.g., the CDR2 sequence of FISGGGTTTYADSVKG (SEQ ID NO: 188). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH7, e.g., the CDR2 sequence of RITGGGSTHYAESVKG (SEQ ID NO: 189).
  • the single domain antibody comprises a CDR2 sequence present in VHH9, e.g., the CDR2 sequence of AISWSGGSTTYADPVKG (SEQ ID NO: 190). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH10, e.g., the CDR2 sequence of AISWSGSSAGYGDSVKG (SEQ ID NO: 191). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH11, e.g., the CDR2 sequence of AIRWSGGRTLYADSVKG (SEQ ID NO: 192).
  • the single domain antibody comprises a CDR2 sequence present in VHH12, e.g., the CDR2 sequence of SITWNGGSTSYADSVKG (SEQ ID NO: 193).
  • the single domain antibody comprises a CDR2 sequence present in VHH1, e.g., the CDR2 sequence of FVAAIDWNGRGTYYRY (SEQ ID NO: 194).
  • the single domain antibody comprises a CDR2 sequence present in VHH2, e.g., the CDR2 sequence of FVAAIDWNGRGTYYRY (SEQ ID NO: 194).
  • the single domain antibody comprises a CDR2 sequence present in VHH3, e.g., the CDR2 sequence of LVARINGGGITH (SEQ ID NO: 195). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH4, e.g., the CDR2 sequence of WVGFIDRIATTT (SEQ ID NO: 196). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH5, e.g., the CDR2 sequence of FVAAITWNGGTTY (SEQ ID NO: 197).
  • the single domain antibody comprises a CDR2 sequence present in VHH6, e.g., the CDR2 sequence of WVAFISGGGTTT (SEQ ID NO: 198). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH7, e.g., the CDR2 sequence of LVARITGGGSTH (SEQ ID NO: 199). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH9, e.g., the CDR2 sequence of FVAAISWSGGSTT (SEQ ID NO: 200).
  • the single domain antibody comprises a CDR2 sequence present in VHH10, e.g., the CDR2 sequence of FVAAISWSGSSAG (SEQ ID NO: 201).
  • the single domain antibody comprises a CDR2 sequence present in VHH11, e.g., the CDR2 sequence of FVAAIRWSGGRTL (SEQ ID NO: 202).
  • the single domain antibody comprises a CDR2 sequence present in VHH12, e.g., the CDR2 sequence of FVASITWNGGSTS (SEQ ID NO: 203).
  • the single domain antibody comprises a CDR2 sequence present in VHH1, e.g., the CDR2 sequence of AIDWNGRGTYYRY (SEQ ID NO: 204). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH2, e.g., the CDR2 sequence of AIDWNGRGTYYRY (SEQ ID NO: 204). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH3, e.g., the CDR2 sequence of RINGGGITH (SEQ ID NO: 205).
  • the single domain antibody comprises a CDR2 sequence present in VHH4, e.g., the CDR2 sequence of FIDRIATTT (SEQ ID NO: 206). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH5, e.g., the CDR2 sequence of AITWNGGTTY (SEQ ID NO: 207). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH6, e.g., the CDR2 sequence of FISGGGTTT (SEQ ID NO: 208).
  • the single domain antibody comprises a CDR2 sequence present in VHH7, e.g., the CDR2 sequence of RITGGGSTH (SEQ ID NO: 209). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH9, e.g., the CDR2 sequence of AISWSGGSTT (SEQ ID NO: 210). In various embodiments of the aspects described herein the single domain antibody comprises a CDR2 sequence present in VHH10, e.g., the CDR2 sequence of AISWSGSSAG (SEQ ID NO: 211).
  • the single domain antibody comprises a CDR2 sequence present in VHH11, e.g., the CDR2 sequence of AIRWSGGRTL (SEQ ID NO: 212).
  • the single domain antibody comprises a CDR2 sequence present in VHH12, e.g., the CDR2 sequence of SITWNGGSTS (SEQ ID NO: 213).
  • the single domain antibody comprises a CDR3 sequence present in VHH1, e.g., the CDR3 sequence of GSIDLNWYGGMDY (SEQ ID NO: 60).
  • the single domain antibody comprises a CDR3 sequence present in VHH2, e.g., the CDR3 sequence of TTVLTDPRVLNEYAT (SEQ ID NO: 61). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH3, e.g., the CDR3 sequence of DVFGSSGYVETY (SEQ ID NO: 62). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH4, e.g., the CDR3 sequence of PLTAR (SEQ ID NO: 63).
  • the single domain antibody comprises a CDR3 sequence present in VHH5, e.g., the CDR3 sequence of DPFNQGY (SEQ ID NO: 64). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH6, e.g., the CDR3 sequence of PLTSR (SEQ ID NO: 65). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH7, e.g., the CDR3 sequence of MVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 66).
  • the single domain antibody comprises a CDR3 sequence present in VHH9, e.g., the CDR3 sequence of DQRGY (SEQ ID NO: 67) or QRGY (SEQ ID NO: 271).
  • the single domain antibody comprises a CDR3 sequence present in VHH10, e.g., the CDR3 sequence of DPFNQGY (SEQ ID NO: 68).
  • the single domain antibody comprises a CDR3 sequence present in VHH11, e.g., the CDR3 sequence of DLAEYSGTYSSPADSPAGYDY (SEQ ID NO: 69).
  • the single domain antibody comprises a CDR3 sequence present in VHH12, e.g., the CDR3 sequence of ARYYVSGTYFPANY (SEQ ID NO: 70).
  • the single domain antibody comprises a CDR3 sequence present in VHH1, e.g., the CDR3 sequence of GSIDLNWYGGMDY (SEQ ID NO: 71) or SIDLNWYGGMD (SEQ ID NO: 272).
  • the single domain antibody comprises a CDR3 sequence present in VHH2, e.g., the CDR3 sequence of TTVLTDPRVLNEYAT (SEQ ID NO: 72) or TVLTDPRVLNEYA (SEQ ID NO: 273).
  • the single domain antibody comprises a CDR3 sequence present in VHH3, e.g., the CDR3 sequence of DVFGSSGYVETY (SEQ ID NO: 73) or VFGSSGYVET (SEQ ID NO: 274).
  • the single domain antibody comprises a CDR3 sequence present in VHH4, e.g., the CDR3 sequence of PLTAR (SEQ ID NO: 74) or LTA (SEQ ID NO: 275).
  • the single domain antibody comprises a CDR3 sequence present in VHH5, e.g., the CDR3 sequence of DPFNQGY (SEQ ID NO: 75) or PFNQG (SEQ ID NO: 276).
  • the single domain antibody comprises a CDR3 sequence present in VHH6, e.g., the CDR3 sequence of PLTSR (SEQ ID NO: 76) or LTS (SEQ ID NO: 277).
  • the single domain antibody comprises a CDR3 sequence present in VHH7, e.g., the CDR3 sequence of MVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 77) or VNPIITAWGTIGVREIPDYD (SEQ ID NO: 278).
  • the single domain antibody comprises a CDR3 sequence present in VHH9, e.g., the CDR3 sequence of DQRGY (SEQ ID NO: 78) or RG (SEQ ID NO: 279).
  • the single domain antibody comprises a CDR3 sequence present in VHH10, e.g., the CDR3 sequence of DPFNQGY (SEQ ID NO: 79) or PFNQG (SEQ ID NO: 280).
  • the single domain antibody comprises a CDR3 sequence present in VHH11, e.g., the CDR3 sequence of DLAEYSGTYSSPADSPAGYDY (SEQ ID NO: 80) or LAEYSGTYSSPADSPAGYD (SEQ ID NO: 281).
  • the single domain antibody comprises a CDR3 sequence present in VHH12, e.g., the CDR3 sequence of ARYYVSGTYFPANY (SEQ ID NO: 81) or RYYVSGTYFPAN (SEQ ID NO: 282).
  • the single domain antibody comprises a CDR3 sequence present in VHH1, e.g., the CDR3 sequence of CAAGSIDLNWYGGMDY (SEQ ID NO: 82) or AAGSIDLNWYGGMDY (SEQ ID NO: 283).
  • the single domain antibody comprises a CDR3 sequence present in VHH2, e.g., the CDR3 sequence of CAATTVLTDPRVLNEYAT (SEQ ID NO: 83) or AATTVLTDPRVLNEYAT (SEQ ID NO: 284).
  • the single domain antibody comprises a CDR3 sequence present in VHH3, e.g., the CDR3 sequence of KADVFGSSGYVETY (SEQ ID NO: 84).
  • the single domain antibody comprises a CDR3 sequence present in VHH4, e.g., the CDR3 sequence of NHPLTAR (SEQ ID NO: 85).
  • the single domain antibody comprises a CDR3 sequence present in VHH5, e.g., the CDR3 sequence of AADPFNQGY (SEQ ID NO: 86). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH6, e.g., the CDR3 sequence of NHPLTSR (SEQ ID NO: 87). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH7, e.g., the CDR3 sequence of ASMVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 88).
  • the single domain antibody comprises a CDR3 sequence present in VHH9, e.g., the CDR3 sequence of NDQRGY (SEQ ID NO: 89). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH10, e.g., the CDR3 sequence of AADPFNQGY (SEQ ID NO: 90). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH11, e.g., the CDR3 sequence of AADLAEYSGTYSSPADSPAGYDY (SEQ ID NO: 91).
  • the single domain antibody comprises a CDR3 sequence present in VHH12, e.g., the CDR3 sequence of AAARYYVSGTYFPANY (SEQ ID NO: 92). [00189] In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH1, e.g., the CDR3 sequence of GSIDLNWYGGMDY (SEQ ID NO: 214). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH2, e.g., the CDR3 sequence of TTVLTDPRVLNEYAT (SEQ ID NO: 215).
  • the single domain antibody comprises a CDR3 sequence present in VHH3, e.g., the CDR3 sequence of DVFGSSGYVETY (SEQ ID NO: 216). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH4, e.g., the CDR3 sequence of PLTAR (SEQ ID NO: 217). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH5, e.g., the CDR3 sequence of DPFNQGY (SEQ ID NO: 218).
  • the single domain antibody comprises a CDR3 sequence present in VHH6, e.g., the CDR3 sequence of PLTSR (SEQ ID NO: 219).
  • the single domain antibody comprises a CDR3 sequence present in VHH7, e.g., the CDR3 sequence of MVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 220).
  • the single domain antibody comprises a CDR3 sequence present in VHH9, e.g., the CDR3 sequence of QRGY (SEQ ID NO: 221).
  • the single domain antibody comprises a CDR3 sequence present in VHH10, e.g., the CDR3 sequence of DPFNQGY (SEQ ID NO: 222).
  • the single domain antibody comprises a CDR3 sequence present in VHH11, e.g., the CDR3 sequence of DLAEYSGTYSSPADSPAGYDY (SEQ ID NO: 223).
  • the single domain antibody comprises a CDR3 sequence present in VHH12, e.g., the CDR3 sequence of ARYYVSGTYFPANY (SEQ ID NO: 224).
  • the single domain antibody comprises a CDR3 sequence present in VHH1, e.g., the CDR3 sequence of AAGSIDLNWYGGMD (SEQ ID NO: 225). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH2, e.g., the CDR3 sequence of AATTVLTDPRVLNEYA (SEQ ID NO: 226). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH3, e.g., the CDR3 sequence of KADVFGSSGYVET (SEQ ID NO: 227).
  • the single domain antibody comprises a CDR3 sequence present in VHH4, e.g., the CDR3 sequence of NHPLTA (SEQ ID NO: 228). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH5, e.g., the CDR3 sequence of AADPFNQG (SEQ ID NO: 229). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH6, e.g., the CDR3 sequence of NHPLTS (SEQ ID NO: 230).
  • the single domain antibody comprises a CDR3 sequence present in VHH7, e.g., the CDR3 sequence of ASMVNPIITAWGTIGVREIPDYD (SEQ ID NO: 231).
  • the single domain antibody comprises a CDR3 sequence present in VHH9, e.g., the CDR3 sequence of NDQRG (SEQ ID NO: 232).
  • the single domain antibody comprises a CDR3 sequence present in VHH10, e.g., the CDR3 sequence of AADPFNQG (SEQ ID NO: 233).
  • the single domain antibody comprises a CDR3 sequence present in VHH11, e.g., the CDR3 sequence of AADLAEYSGTYSSPADSPAGYD (SEQ ID NO: 234).
  • the single domain antibody comprises a CDR3 sequence present in VHH12, e.g., the CDR3 sequence of AAARYYVSGTYFPAN (SEQ ID NO: 235).
  • the single domain antibody comprises a CDR3 sequence present in VHH1, e.g., the CDR3 sequence of GSIDLNWYGGMDY (SEQ ID NO: 236).
  • the single domain antibody comprises a CDR3 sequence present in VHH2, e.g., the CDR3 sequence of TTVLTDPRVLNEYAT (SEQ ID NO: 237). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH3, e.g., the CDR3 sequence of DVFGSSGYVETY (SEQ ID NO: 238). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH4, e.g., the CDR3 sequence of PLTAR (SEQ ID NO: 239).
  • the single domain antibody comprises a CDR3 sequence present in VHH5, e.g., the CDR3 sequence of DPFNQGY (SEQ ID NO: 240). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH6, e.g., the CDR3 sequence of PLTSR (SEQ ID NO: 241). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH7, e.g., the CDR3 sequence of MVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 242).
  • the single domain antibody comprises a CDR3 sequence present in VHH9, e.g., the CDR3 sequence of QRGY (SEQ ID NO: 243). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH10, e.g., the CDR3 sequence of DPFNQGY (SEQ ID NO: 244). In various embodiments of the aspects described herein the single domain antibody comprises a CDR3 sequence present in VHH11, e.g., the CDR3 sequence of DLAEYSGTYSSPADSPAGYDY (SEQ ID NO: 245).
  • the single domain antibody comprises a CDR3 sequence present in VHH12, e.g., the CDR3 sequence of ARYYVSGTYFPANY (SEQ ID NO: 246).
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: a) VHH2: i) the CDR1 sequence of SYRMG (SEQ ID NO: 1), the CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 30), and the CDR3 sequence of TTVLTDPRVLNEYAT (SEQ ID NO: 61); ii) the CDR1 sequence of GLTFSSY (SEQ ID NO: 10), the CDR2 sequence of DWNGRGTYY (SEQ ID NO: 40) or WNGRGTY (SEQ ID NO: 260), and the CDR3 sequence of TTVLT
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of SYRMG (SEQ ID NO: 1), the CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 30), and the CDR3 sequence of GSIDLNWYGGMDY (SEQ ID NO: 60); ii) the CDR1 sequence of GLTFSSY (SEQ ID NO: 10), the CDR2 sequence of DWNGRGTYY (SEQ ID NO: 40) or WNGRGTY (SEQ ID NO: 260), and the CDR3 sequence of GSIDLNWYGGMDY (SEQ ID NO: 71) or SIDLNWYGGMD (SEQ ID NO: 272); iii) the CDR1 sequence of GLTFSSYR (SEQ ID NO: 20), the CDR2 sequence of I
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of SYRMG (SEQ ID NO: 1), the CDR2 sequence of AIDWNGRGTYYRYYADSVKG (SEQ ID NO: 30), and the CDR3 sequence of TTVLTDPRVLNEYAT (SEQ ID NO: 61); ii) the CDR1 sequence of GLTFSSY (SEQ ID NO: 10), the CDR2 sequence of DWNGRGTYY (SEQ ID NO: 40) or WNGRGTY (SEQ ID NO: 260), and the CDR3 sequence of TTVLTDPRVLNEYAT (SEQ ID NO: 72) or TVLTDPRVLNEYA (SEQ ID NO: 273); iii) the CDR1 sequence of GLTFSSYR (SEQ ID NO: 20), the CDR2 sequence of the single domain antibody selected from the group
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of INVMG (SEQ ID NO: 2), the CDR2 sequence of RINGGGITHYAESVKG (SEQ ID NO: 31), and the CDR3 sequence of DVFGSSGYVETY (SEQ ID NO: 62); ii) the CDR1 sequence of GSIFSIN (SEQ ID NO: 11), the CDR2 sequence of NGGGI (SEQ ID NO: 41) or GGG (SEQ ID NO: 261), and the CDR3 sequence of DVFGSSGYVETY (SEQ ID NO: 73) or VFGSSGYVET (SEQ ID NO: 274); iii) the CDR1 sequence of GSIFSINV (SEQ ID NO: 21), the CDR2 sequence of INGGGIT (SEQ ID NO: 51), and the CDR
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of SNAMG (SEQ ID NO: 3), the CDR2 sequence of FIDRIATTTIATSVKG (SEQ ID NO: 32), and the CDR3 sequence of PLTAR (SEQ ID NO: 63); ii) the CDR1 sequence of GTSVSSN (SEQ ID NO: 12), the CDR2 sequence of DRIAT (SEQ ID NO: 42) or RIA (SEQ ID NO: 262), and the CDR3 sequence of PLTAR (SEQ ID NO: 74) or LTA (SEQ ID NO: 275); iii) the CDR1 sequence of GTSVSSNA (SEQ ID NO: 22), the CDR2 sequence of IDRIATT (SEQ ID NO: 52), and the CDR3 sequence of NHPLTAR (SEQ ID NO: 85); i
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of SYAMG (SEQ ID NO: 4), the CDR2 sequence of AITWNGGTTYYADSVKG (SEQ ID NO: 33), and the CDR3 sequence of DPFNQGY (SEQ ID NO: 64); ii) the CDR1 sequence of GRTFSSY (SEQ ID NO: 13), the CDR2 sequence of TWNGGT (SEQ ID NO: 43) or WNGG (SEQ ID NO: 263), and the CDR3 sequence of DPFNQGY (SEQ ID NO: 75) or PFNQG (SEQ ID NO: 276); iii) the CDR1 sequence of GRTFSSYA (SEQ ID NO: 23), the CDR2 sequence of ITWNGGTT (SEQ ID NO: 53), and the CDR3 sequence of the single domain antibody selected from the group consist
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of SDAMG (SEQ ID NO: 5), the CDR2 sequence of FISGGGTTTYADSVKG (SEQ ID NO: 34), and the CDR3 sequence of PLTSR (SEQ ID NO: 65); ii) the CDR1 sequence of GSSVSSD (SEQ ID NO: 14), the CDR2 sequence of SGGGT (SEQ ID NO: 44) or GGG (SEQ ID NO: 264), and the CDR3 sequence of PLTSR (SEQ ID NO: 76) or LTS (SEQ ID NO: 277); iii) the CDR1 sequence of GSSVSSDA (SEQ ID NO: 24), the CDR2 sequence of ISGGGTT (SEQ ID NO: 54), and the CDR3 sequence of NHPLTSR (SEQ ID NO: 87
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of INVMG (SEQ ID NO: 6), the CDR2 sequence of RITGGGSTHYAESVKG (SEQ ID NO: 35), and the CDR3 sequence of MVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 66); ii) the CDR1 sequence of RSIGSIN (SEQ ID NO: 15), the CDR2 sequence of TGGGS (SEQ ID NO: 45) or GGG (SEQ ID NO: 265), and the CDR3 sequence of MVNPIITAWGTIGVREIPDYDY (SEQ ID NO: 77) or VNPIITAWGTIGVREIPDYD (SEQ ID NO: 278); iii) the CDR1 sequence of RSIGSINV (SEQ ID NO: 25), the CDR2 sequence of I
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of TYRMG (SEQ ID NO: 7), the CDR2 sequence of AISWSGGSTTYADPVKG (SEQ ID NO: 36), and the CDR3 sequence of DQRGY (SEQ ID NO: 67) or QRGY (SEQ ID NO: 271); ii) the CDR1 sequence of GRTFSTY (SEQ ID NO: 16), the CDR2 sequence of SWSGGS (SEQ ID NO: 46) or WSGG (SEQ ID NO: 266), and the CDR3 sequence of DQRGY (SEQ ID NO: 78) or RG (SEQ ID NO: 279); iii) the CDR1 sequence of GRTFSTYR (SEQ ID NO: 26), the CDR2 sequence of ISWSGGST (SEQ ID NO: 56),
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of RYAMG (SEQ ID NO: 8), the CDR2 sequence of AISWSGSSAGYGDSVKG (SEQ ID NO: 37), and the CDR3 sequence of DPFNQGY (SEQ ID NO: 68); ii) the CDR1 sequence of GFTFTRY (SEQ ID NO: 17), the CDR2 sequence of SWSGSS (SEQ ID NO: 47) or WSGS (SEQ ID NO: 267), and the CDR3 sequence of DPFNQGY (SEQ ID NO: 79) or PFNQG (SEQ ID NO: 280); iii) the CDR1 sequence of GFTFTRYA (SEQ ID NO: 27), the CDR2 sequence of ISWSGSSA (SEQ ID NO: 57), and the CDR3 sequence of the single domain antibody selected from the group
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of FTTYRMG (SEQ ID NO: 258) or TYRMG (SEQ ID NO: 259), the CDR2 sequence of AIRWSGGRTLYADSVKG (SEQ ID NO: 38), and the CDR3 sequence of DLAEYSGTYSSPADSPAGYDY (SEQ ID NO: 69); ii) the CDR1 sequence of GRTFTTY (SEQ ID NO: 18), the CDR2 sequence of RWSGGR (SEQ ID NO: 48) or WSGG (SEQ ID NO: 268), and the CDR3 sequence of DLAEYSGTYSSPADSPAGYDY (SEQ ID NO: 80) or LAEYSGTYSSPADSPAGYD (SEQ ID NO: 281); iii) the CDR1 sequence of GRT
  • the single domain antibody provided herein comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of the single domain antibody selected from the group consisting of: i) the CDR1 sequence of FNTYAMG (SEQ ID NO: 9), the CDR2 sequence of SITWNGGSTSYADSVKG (SEQ ID NO: 39), and the CDR3 sequence of ARYYVSGTYFPANY (SEQ ID NO: 70); ii) the CDR1 sequence of GRTLSFNTY (SEQ ID NO: 19), the CDR2 sequence of TWNGGS (SEQ ID NO: 49) or WNGG (SEQ ID NO: 269), and the CDR3 sequence of ARYYVSGTYFPANY (SEQ ID NO: 81) or RYYVSGTYFPAN (SEQ ID NO: 282); iii) the CDR1 sequence of GRTLSFNTYA (SEQ ID NO: 29), the CDR2 sequence of ITWNGGST (SEQ ID NO:
  • the single domain antibody provided herein has one or more CDR regions from VHH1.
  • the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 93.
  • the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 93.
  • the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 93.
  • the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 93.
  • the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 93. CDR sequences can be determined according to well-known numbering systmes.
  • CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems.
  • the residues from each of these hypervariable regions or CDRs are noted in Table 1 above.
  • the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering.
  • the CDRs are according to Chothia numbering.
  • the CDRs are according to Contact numbering.
  • the CDRs are according to IMGT numbering.
  • the single domain antibody provided herein has one or more CDR regions from VHH2.
  • the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 94. In other embodiments, the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 94.
  • the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 94. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering.
  • the single domain antibody provided herein has one or more CDR regions from VHH3. [00209] In some embodiments, the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 95.
  • the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 95.
  • the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 95.
  • CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering. [00210] In some embodiments, the single domain antibody provided herein has one or more CDR regions from VHH4.
  • the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 96. In other embodiments, the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 96.
  • the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 96. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering.
  • the single domain antibody provided herein has one or more CDR regions from VHH5. [00213] In some embodiments, the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 97.
  • the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 97.
  • the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 97.
  • CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering. [00214] In some embodiments, the single domain antibody provided herein has one or more CDR regions from VHH6.
  • the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 98. In other embodiments, the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 98.
  • the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 98. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering.
  • the single domain antibody provided herein has one or more CDR regions from VHH7. [00217] In some embodiments, the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 99.
  • the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 99.
  • the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 99.
  • CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering. [00218] In some embodiments, the single domain antibody provided herein has one or more CDR regions from VHH9.
  • the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 100. In other embodiments, the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 100.
  • the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 100. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering. [00220] In some embodiments, the single domain antibody provided herein has one or more CDR regions from VHH10. [00221] In some embodiments, the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 101.
  • the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 101.
  • the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 101.
  • CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering.
  • the single domain antibody that binds to pIgR provided herein has one or more CDR regions from VHH11.
  • the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 102.
  • the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 102.
  • the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 102.
  • the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 102.
  • the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 102.
  • CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering.
  • the single domain antibody that binds to pIgR provided herein has one or more CDR regions from VHH12.
  • the single domain antibody has a CDR1 having an amino acid sequence of the CDR1 as set forth in SEQ ID NO: 103.
  • the single domain antibody has a CDR2 having an amino acid sequence of the CDR2 as set forth in SEQ ID NO: 103.
  • the single domain antibody has a CDR3 having an amino acid sequence of the CDR3 as set forth in SEQ ID NO: 103.
  • the single domain antibody has a CDR1 and a CDR2 having amino acid sequences of the CDR1 and the CDR2 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having amino acid sequences of the CDR1 and the CDR3 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having amino acid sequences of the CDR2 and the CDR3 as set forth in SEQ ID NO: 103.
  • the single domain antibody has a CDR1, a CDR2, and a CDR3 having amino acid sequences of the CDR1, the CDR2, and the CDR3 as set forth in SEQ ID NO: 103.
  • CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDRs are according to IMGT numbering.
  • the single domain antibody further comprises one or more framework regions of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH7, VHH9, VHH10, VHH11 and VHH12.
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of QVQLVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGSIDLNWYGGMD YWGQGTQVTVSS (SEQ ID NO: 93).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of EVQVVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTVYLQMNSLKPEDTAVYYCAATTVLTDPRVLNEYA TWGQGTQVTVSS (SEQ ID NO: 94).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of QLQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVARINGGGITHY AESVKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCKADVFGSSGYVETYWGQGTQV TVSS (SEQ ID NO: 95).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of EVQVVESGGGLVQAGGSLRLSCAVSGTSVSSNAMGWYRQAPGKQREWVGFIDRIATTT IATSVKGRFAITRDNAKNTVYLQMSGLKPEDTAVYYCNHPLTARWGQGTQVTVSS (SEQ ID NO: 96).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWNGGTT YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPFNQGYWGQGTQVTVS S (SEQ ID NO: 97).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of EVQLVESGGGLVQAGGSLRLSCAVSGSSVSSDAMGWYRQAPGNQRAWVAFISGGGTT TYADSVKGRFTISRDNTKNTVYLHMNSLKPEDTAVYYCNHPLTSRWGQGTQVTVSS (SEQ ID NO: 98).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of EVQVVESGGGLVQAGGSLRLACVASRSIGSINVMGWYRQAPGKQRDLVARITGGGSTH YAESVKGRFTISRDNAKNTVYLQMNSLEPEDTAVYYCASMVNPIITAWGTIGVREIPDY DYWGQGTQVTVSS (SEQ ID NO: 99).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of QVQLVESGGGLVQAGGSLRLSCAVSGRTFSTYRMGWFRQAPGKERSFVAAISWSGGST TYADPVKGRFTISRDNAKNTVYLRMNSLKPEDTAVYYCNDQRGYWGQGTLVTVSS (SEQ ID NO: 100).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of EVQVVESGGGLVQAGGSLRLSCAASGFTFTRYAMGWFRQAPGKERSFVAAISWSGSSA GYGDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADPFNQGYWGQGTQVTVS S (SEQ ID NO: 101).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of EVQVVESGGGLVQAGGSLRLSCAASGRTFTTYRMGWFRQAPGKEREFVAAIRWSGGRT LYADSVKGRFTISRDNAKNTAYLQMNNLRPEDTAVYYCAADLAEYSGTYSSPADSPAG YDYWGQGTQVTVSS (SEQ ID NO: 102).
  • the single domain antibody comprises a framework derived from a VHH domain comprising the sequence of QVQLVETGGGLVQAGDSLRLSCAASGRTLSFNTYAMGWFRQAPGKEREFVASITWNG GSTSYADSVKGRFTITRDNAKNTATLRMNSLQPDDTAVYYCAAARYYVSGTYFPANY WGQGTQVTVSS (SEQ ID NO: 103).
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 93.
  • the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 93.
  • the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 93.
  • the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 93.
  • the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 93. In some embodiments, the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 93.
  • the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 93.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 94.
  • the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 94.
  • the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 94.
  • the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 94.
  • the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 94.
  • the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 94. In some embodiments, the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 94. In a specific embodiment, the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 94.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 95.
  • the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 95.
  • the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 95.
  • the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 95. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 95.
  • the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 95.
  • the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 95.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 96.
  • the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 96.
  • the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 96.
  • the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 96.
  • the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 96.
  • the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 96.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 97.
  • the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 97.
  • the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 97.
  • the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 97.
  • the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 97.
  • the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 97. In a specific embodiment, the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 97.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 98.
  • the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 98.
  • the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 98.
  • the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 98. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 98.
  • the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 98.
  • the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 98.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 99.
  • the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 99.
  • the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 99.
  • the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 99.
  • the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 99. In some embodiments, the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 99.
  • the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 99.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 100.
  • the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 100.
  • the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 100.
  • the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 100.
  • the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 100.
  • the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 100.
  • the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 100.
  • the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 100.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 101.
  • the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 101.
  • the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 101.
  • the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 101.
  • the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 101.
  • the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 101.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 102.
  • the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 102.
  • the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 102.
  • the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 102.
  • the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 102.
  • the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 102. In some embodiments, the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 102. In a specific embodiment, the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 102.
  • the single domain antibody provided herein comprises a FR1 having an amino acid sequence of the FR1 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody provided herein comprises a FR2 having an amino acid sequence of the FR2 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody provided herein comprises a FR3 having an amino acid sequence of the FR3 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody provided herein comprises a FR4 having an amino acid sequence of the FR4 as set forth in SEQ ID NO: 103.
  • the single domain antibody provided herein comprises a FR1 and a FR2 having amino acid sequences of the FR1 and the FR2 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR3 having amino acid sequences of the FR1 and the FR3 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody provided herein comprises a FR1 and a FR4 having amino acid sequences of the FR1 and the FR4 as set forth in SEQ ID NO: 103.
  • the single domain antibody provided herein comprises a FR2 and a FR3 having amino acid sequences of the FR2 and the FR3 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody provided herein comprises a FR2 and a FR4 having amino acid sequences of the FR2 and the FR4 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody provided herein comprises a FR3 and a FR4 having amino acid sequences of the FR3 and the FR24 as set forth in SEQ ID NO: 103.
  • the single domain antibody provided herein comprises a FR1, a FR2, and a FR3 having amino acid sequences of the FR1, the FR2, and the FR3 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR2, and a FR4 having amino acid sequences of the FR1, the FR2, and the FR4 as set forth in SEQ ID NO: 103. In some embodiments, the single domain antibody provided herein comprises a FR1, a FR3, and a FR4 having amino acid sequences of the FR1, the FR3, and the FR4 as set forth in SEQ ID NO: 103.
  • the single domain antibody provided herein comprises a FR2, a FR3, and a FR4 having amino acid sequences of the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 103.
  • the single domain antibody provided herein comprises a FR1, a FR2, a FR3, and a FR4 having amino acid sequences of the FR1, the FR2, the FR3, and the FR4 as set forth in SEQ ID NO: 103.
  • the single domain antibody provided herein is a humanized single domain antibody.
  • Framework regions described herein are determined based upon the boundaries of the CDR numbering system.
  • the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format, from the N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4.
  • FR1 is defined as the amino acid residues N-terminal to the CDR1 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system
  • FR2 is defined as the amino acid residues between CDR1 and CDR2 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system
  • FR3 is defined as the amino acid residues between CDR2 and CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system
  • FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.
  • the single domain antibody comprises a sequence of QVQLVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGSIDLNWYGGMD YWGQGTQVTVSS (SEQ ID NO: 93).
  • the single domain antibody comprises a sequence of EVQVVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTVYLQMNSLKPEDTAVYYCAATTVLTDPRVLNEYA TWGQGTQVTVSS (SEQ ID NO: 94).
  • the single domain antibody comprises a sequence of QLQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVARINGGGITHY AESVKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCKADVFGSSGYVETYWGQGTQV TVSS (SEQ ID NO: 95).
  • the single domain antibody comprises a sequence of EVQVVESGGGLVQAGGSLRLSCAVSGTSVSSNAMGWYRQAPGKQREWVGFIDRIATTT IATSVKGRFAITRDNAKNTVYLQMSGLKPEDTAVYYCNHPLTARWGQGTQVTVSS (SEQ ID NO: 96).
  • the single domain antibody comprises a sequence of QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWNGGTT YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPFNQGYWGQGTQVTVS S (SEQ ID NO: 97).
  • the single domain antibody comprises a sequence of EVQLVESGGGLVQAGGSLRLSCAVSGSSVSSDAMGWYRQAPGNQRAWVAFISGGGTT TYADSVKGRFTISRDNTKNTVYLHMNSLKPEDTAVYYCNHPLTSRWGQGTQVTVSS (SEQ ID NO: 98).
  • the single domain antibody comprises a sequence of EVQVVESGGGLVQAGGSLRLACVASRSIGSINVMGWYRQAPGKQRDLVARITGGGSTH YAESVKGRFTISRDNAKNTVYLQMNSLEPEDTAVYYCASMVNPIITAWGTIGVREIPDY DYWGQGTQVTVSS (SEQ ID NO: 99).
  • the single domain antibody comprises a sequence of QVQLVESGGGLVQAGGSLRLSCAVSGRTFSTYRMGWFRQAPGKERSFVAAISWSGGST TYADPVKGRFTISRDNAKNTVYLRMNSLKPEDTAVYYCNDQRGYWGQGTLVTVSS (SEQ ID NO: 100).
  • the single domain antibody comprises a sequence of EVQVVESGGGLVQAGGSLRLSCAASGFTFTRYAMGWFRQAPGKERSFVAAISWSGSSA GYGDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADPFNQGYWGQGTQVTVS S (SEQ ID NO: 101).
  • the single domain antibody comprises a sequence of EVQVVESGGGLVQAGGSLRLSCAASGRTFTTYRMGWFRQAPGKEREFVAAIRWSGGRT LYADSVKGRFTISRDNAKNTAYLQMNNLRPEDTAVYYCAADLAEYSGTYSSPADSPAG YDYWGQGTQVTVSS (SEQ ID NO: 102).
  • the single domain antibody comprises a sequence of QVQLVETGGGLVQAGDSLRLSCAASGRTLSFNTYAMGWFRQAPGKEREFVASITWNG GSTSYADSVKGRFTITRDNAKNTATLRMNSLQPDDTAVYYCAAARYYVSGTYFPANY WGQGTQVTVSS (SEQ ID NO: 103).
  • an antibody described herein or an antigen-binding fragment thereof comprises amino acid sequences with certain percent identity relative to any one of antibodies VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH7, VHH9, VHH10, VHH11 and VHH12.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A.87:2264 2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:58735877.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol.215:403.
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res.25:33893402.
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Altschul BLAST
  • Gapped BLAST Altschul BLAST
  • PSI Blast programs the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).
  • NCBI National Center for Biotechnology Information
  • Another preferred, non limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:1117. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 93, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 94, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 95, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 96, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 97, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 98, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 99, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 100, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 101, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 102, wherein the single domain antibody binds to pIgR.
  • the single domain antibody described herein comprises a VHH domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 103, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 93, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 94, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 95, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 96, wherein the single domain antibody immunospecifically binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 97, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 98, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 99, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 100, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 101, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 102, wherein the single domain antibody binds to pIgR.
  • the single domain antibody provided herein comprises a framework having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 103, wherein the single domain antibody binds to pIgR. 5.2.2. Humanized Single Domain Antibodies [00287]
  • the single domain antibodies described herein include humanized single domain antibodies. General strategies to humanize single domain antibodies from Camelidae species have been described (see, e.g., Vincke et al., J. Biol.
  • Humanized antibodies such as the humanized single domain antibodies disclosed herein can also be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International publication No. WO 91/09967; and U.S.
  • Patent Nos.5,225,539, 5,530,101, and 5,585,089) veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Patent No.5,565,332), and techniques disclosed in, e.g., U.S. Pat. No.6,407,213, U.S. Pat. No.5,766,886, WO 9317105, Tan et al., J. Immunol.
  • single domain antibodies provided herein can be humanized single domain antibodies that bind to pIgR, including human pIgR.
  • humanized single chain antibodies of the present disclosure may comprise one or more CDRs of VHH1, VHH2, VHH3, VHH4, VHH5, VHH6, VHH7, VHH9, VHH10, VHH11 and/or VHH12.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human.
  • non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Humanization may be performed, for example, following the method of Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the CDRs of the parent non-human antibody are grafted onto a human antibody framework. For example, Padlan et al.
  • the human sequence that is closest to that of the non-human antibody may be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol.151:2296-308; and Chothia et al., 1987, J. Mol. Biol. 196:901-17).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; and Presta et al., 1993, J. Immunol.151:2623-32).
  • the framework is derived from the consensus sequences of the most abundant human subclasses, VL ⁇ subgroup I (VL ⁇ I) and VH subgroup III (VHIII).
  • human germline genes are used as the source of the framework regions.
  • FR homology is irrelevant. The method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors.
  • antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • HSC Human String Content
  • Antibody variants may be isolated from phage, ribosome, and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol.23:1105-16; Dufner et al., 2006, Trends Biotechnol.24:523-29; Feldhaus et al., 2003, Nat. Biotechnol.21:163-70; and Schlapschy et al., 2004, Protein Eng. Des. Sel.17:847-60).
  • residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol.224:487-99), or from the more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem.272:10678-84).
  • FR shuffling whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall’Acqua et al., 2005, Methods 36:43-60).
  • a one-step FR shuffling process may be used. Such a process has been shown to be efficient, as the resulting antibodies exhibited improved biochemical and physicochemical properties including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol.44:3049-60).
  • the “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. This methodology typically results in epitope retention and identification of antibodies from multiple subclasses with distinct human V-segment CDRs.
  • the “human engineering” method involves altering a non-human antibody or antibody fragment by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies.
  • the technique involves classifying amino acid residues of a non-human antibody as “low risk,” “moderate risk,” or “high risk” residues.
  • the classification is performed using a global risk/reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody’s folding.
  • the particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody’s variable regions with the corresponding region of a specific or consensus human antibody sequence.
  • a composite human antibody can be generated using, for example, Composite Human AntibodyTM technology (Antitope Ltd., Cambridge, United Kingdom).
  • variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody.
  • a deimmunized antibody is an antibody in which T-cell epitopes have been removed. Methods for making deimmunized antibodies have been described. See, e.g., Jones et al., Methods Mol Biol.2009;525:405-23, xiv, and De Groot et al., Cell. Immunol.244:148- 153(2006)).
  • Deimmunized antibodies comprise T-cell epitope-depleted variable regions and human constant regions.
  • variable regions of an antibody are cloned and T-cell epitopes are subsequently identified by testing overlapping peptides derived from the variable regions of the antibody in a T cell proliferation assay.
  • T cell epitopes are identified via in silico methods to identify peptide binding to human MHC class II. Mutations are introduced in the variable regions to abrogate binding to human MHC class II. Mutated variable regions are then utilized to generate the deimmunized antibody. 5.2.3.
  • Single Domain Antibody Variants [00302] In some embodiments, amino acid sequence modification(s) of the single domain antibodies that bind to pIgR described herein are contemplated.
  • single domain antibody variants can be prepared.
  • single domain antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art who appreciate that amino acid changes may alter post-translational processes of the single domain antibody.
  • the single domain antibodies provided herein are chemically modified, for example, by the covalent attachment of any type of molecule to the single domain antibody.
  • the antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or conjugation to one or more immunoglobulin domains (e.g., Fc or a portion of an Fc). Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc.
  • the antibody may contain one or more non-classical amino acids.
  • Variations may be a substitution, deletion, or insertion of one or more codons encoding the single domain antibody or polypeptide that results in a change in the amino acid sequence as compared with the original antibody or polypeptide.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule.
  • the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues.
  • the variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the parental antibodies.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue.
  • Single domain antibodies generated by conservative amino acid substitutions are included in the present disclosure.
  • an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
  • families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g.
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed and the activity of the protein can be determined. conservative (e.g., within an amino acid group with similar properties and/or side chains) substitutions may be made, so as to maintain or not significantly change the properties.
  • Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed.1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His(H).
  • Naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • any cysteine residue not involved in maintaining the proper conformation of the single domain antibody also may be substituted, for example, with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and to prevent aberrant crosslinking.
  • the variations can be made using methods known in the art such as oligonucleotide- mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis see, e.g., Carter, 1986, Biochem J.237:1-7; and Zoller et al., 1982, Nucl. Acids Res.10:6487-500
  • cassette mutagenesis see, e.g., Wells et al., 1985, Gene 34:315-23
  • other known techniques can be performed on the cloned DNA to produce the single domain antibody variant DNA. 5.2.4.
  • antibody variants having an improved property such as affinity, stability, or expression level as compared to a parent antibody may be prepared by in vitro affinity maturation.
  • in vitro affinity maturation is based on the principles of mutation and selection.
  • Libraries of antibodies are displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cell) or in association (e.g., covalently or non-covalently) with their encoding mRNA or DNA.
  • Affinity selection of the displayed antibodies allows isolation of organisms or complexes carrying the genetic information encoding the antibodies.
  • Phage display is a widespread method for display and selection of antibodies. The antibodies are displayed on the surface of Fd or M13 bacteriophages as fusions to the bacteriophage coat protein. Selection involves exposure to antigen to allow phage-displayed antibodies to bind their targets, a process referred to as “panning.” Phage bound to antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For review, see, for example, Hoogenboom, 2002, Methods. Mol.
  • the antibody may be fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall through disulfide bonds to Aga1p. Display of a protein via Aga2p projects the protein away from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall.
  • Magnetic separation and flow cytometry are used to screen the library to select for antibodies with improved affinity or stability. Binding to a soluble antigen of interest is determined by labeling of yeast with biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Variations in surface expression of the antibody can be measured through immunofluorescence labeling of either the hemagglutinin or c-Myc epitope tag flanking the single chain antibody (e.g., scFv). Expression has been shown to correlate with the stability of the displayed protein, and thus antibodies can be selected for improved stability as well as affinity (see, e.g., Shusta et al., 1999, J. Mol. Biol.292:949-56).
  • yeast display An additional advantage of yeast display is that displayed proteins are folded in the endoplasmic reticulum of the eukaryotic yeast cells, taking advantage of endoplasmic reticulum chaperones and quality-control machinery. Once maturation is complete, antibody affinity can be conveniently “titrated” while displayed on the surface of the yeast, eliminating the need for expression and purification of each clone.
  • a theoretical limitation of yeast surface display is the potentially smaller functional library size than that of other display methods; however, a recent approach uses the yeast cells’ mating system to create combinatorial diversity estimated to be 10 14 in size (see, e.g., U.S. Pat. Publication 2003/0186374; and Blaise et al., 2004, Gene 342:211–18).
  • antibody-ribosome-mRNA (ARM) complexes are generated for selection in a cell-free system.
  • the DNA library coding for a particular library of antibodies is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold.
  • the resulting complex of mRNA, ribosome, and protein can bind to surface-bound ligand, allowing simultaneous isolation of the antibody and its encoding mRNA through affinity capture with the ligand.
  • ribosome- bound mRNA is then reverse transcribed back into cDNA, which can then undergo mutagenesis and be used in the next round of selection (see, e.g., Fukuda et al., 2006, Nucleic Acids Res. 34:e127).
  • mRNA display a covalent bond between antibody and mRNA is established using puromycin as an adaptor molecule (Wilson et al., 2001, Proc. Natl. Acad. Sci. USA 98:3750-55).
  • the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube.
  • random mutations can be introduced easily after each selection round, for example, by non-proofreading polymerases, as no library must be transformed after any diversification step.
  • mammalian display systems may be used.
  • Diversity may also be introduced into the CDRs of the antibody libraries in a targeted manner or via random introduction. The former approach includes sequentially targeting all the CDRs of an antibody via a high or low level of mutagenesis or targeting isolated hot spots of somatic hypermutations (see, e.g., Ho et al., 2005, J. Biol.
  • Chem.280:607-17 residues suspected of affecting affinity on experimental basis or structural reasons.
  • Diversity may also be introduced by replacement of regions that are naturally diverse via DNA shuffling or similar techniques (see, e.g., Lu et al., 2003, J. Biol. Chem.278:43496-507; U.S. Pat. Nos.5,565,332 and 6,989,250).
  • Alternative techniques target hypervariable loops extending into framework- region residues (see, e.g., Bond et al., 2005, J. Mol. Biol.348:699-709) employ loop deletions and insertions in CDRs or use hybridization-based diversification (see, e.g., U.S. Pat.
  • single domain antibodies can be immobilized onto solid supports, columns, pins, or cellulose/poly(vinylidene fluoride) membranes/other filters, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin- coated beads or used in any other method for panning display libraries.
  • Hoogenboom 2005, Nature Biotechnology 23:1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4:39- 51; and references therein. 5.2.5. Modifications of Single Domain Antibodies
  • Covalent modifications of single domain antibodies are included within the scope of the present disclosure.
  • Covalent modifications include reacting targeted amino acid residues of a single domain antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the single domain antibody.
  • Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
  • the single domain antibody that binds to pIgR of the disclosure may also be genetically fused or conjugated to one or more immunoglobulin constant regions or portions thereof (e.g., Fc) to extend half-life and/or to impart known Fc- mediated effector functions.
  • immunoglobulin constant regions or portions thereof e.g., Fc
  • the single chain antibody that binds to pIgR of the present disclosure may also be modified to form chimeric molecules comprising the single chain antibody that binds to pIgR fused to another, heterologous polypeptide or amino acid sequence, for example, an epitope tag (see, e.g., Terpe, 2003, Appl. Microbiol. Biotechnol.60:523-33) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).
  • the single chain antibody that binds to pIgR may also be used to generate pIgR binding chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • fusion proteins comprising the single chain antibody that binds to pIgR of the disclosure and a heterologous polypeptide.
  • the heterologous polypeptide to which the antibody is genetically fused or chemically conjugated is useful for targeting the antibody to cells having cell surface-expressed pIgR.
  • panels of antibodies that bind to an pIgR antigen are also provided herein.
  • the panels of antibodies have different association rates, different dissociation rates, different affinities for an pIgR antigen, and/or different specificities for an pIgR antigen.
  • the panels comprise or consist of about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 antibodies or more.
  • Panels of antibodies can be used, for example, in 96-well or 384-well plates, for assays such as ELISAs. 5.2.6.
  • Single domain antibodies may be produced by culturing cells transformed or transfected with a vector containing a single domain antibody-encoding nucleic acids.
  • Polynucleotide sequences encoding polypeptide components of the antibody of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridomas cells or B cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques.
  • sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells.
  • a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells.
  • Many vectors that are available and known in the art can be used for the purpose of the present disclosure. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector.
  • Host cells suitable for expressing antibodies of the present disclosure include prokaryotes such as Archaebacteria and Eubacteria, including Gram-negative or Gram-positive organisms, eukaryotic microbes such as filamentous fungi or yeast, invertebrate cells such as insect or plant cells, and vertebrate cells such as mammalian host cell lines.
  • Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Antibodies produced by the host cells are purified using standard protein purification methods as known in the art.
  • anti- pIgR antibodies may be prepared by alternative methods, which are well known in the art.
  • the appropriate amino acid sequence, or portions thereof may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis (1969); and Merrifield, 1963, J. Am. Chem. Soc.85:2149-54).
  • In vitro protein synthesis may be performed using manual techniques or by automation.
  • Various portions of the anti- pIgR antibody may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti- pIgR antibody.
  • antibodies may be purified from cells or bodily fluids, such as milk, of a transgenic animal engineered to express the antibody, as disclosed, for example, in U.S. Pat. Nos.5,545,807 and 5,827,690.
  • the single domain antibodies, or other pIgR binders can be generated by immunizing llamas using mpIgR and hpIgR extracellular domain (ECD), performing single B- cell sorting, undertaking V-gene extraction, cloning the pIgR binders, such as VHH -Fc fusions, and then performing small scale expression and purification.
  • Additional screening of the single domain antibodies and other molecules that bind to pIgR can be performed, including one or more of selecting for ELISA-positive, BLI-positive, and K D less than 100 nM. These selection criteria can be combined as shown in Figure 8 (VHH generated from mpIgR antigen) and Figure 9 (VHH generated from hpIgR antigen). Additionally, individual VHH binders (and other molecules that bind to pIgR) can be assayed for their ability to bind to MDCK cells expressing pIgR, e.g., hpIgR.
  • Such assay can be performed using FACS analysis with MDCK cells expressing hpIgR, and measuring the mean fluorescence intensity (MFI) of fluorescently-labeled VHH molecules. The results of such experiment are shown in Figure 10. The staining of hpIgR on a monolayer of MDCK cells is shown in Figure 11. 5.3.
  • Therapeutic Molecules Comprising the Single Domain Antibodies [00328] In one aspect, provided herein is a therapeutic molecule comprising a single domain antibody (e.g., a VHH domain) provided herein and a therapeutic agent. [00329] In various embodiments, the single domain antibody provided herein can be genetically fused or chemically conjugated to any agents for delivery of these agents, for example, protein- based entities.
  • the single domain antibody may be chemically-conjugated to the agent, or otherwise non-covalently conjugated to the agent.
  • the single domain antibodies provided herein are useful for delivering agents that can be used to treat subjects, such as biologics (including protein based therapeutics such as peptides and antibodies), and nucleotide based therapeutics such as viral gene therapeutics or RNA therapeutics).
  • the agent can be a diabetes medication, optionally selected from a group consisting of insulin, glucagon-like-peptide-1, insulin-mimic peptides, and glucagon-like- peptide-1-mimic peptides.
  • the agent can be a peptide or antibody (or a fragment thereof), optionally selected from a group consisting of an anti-TNF-alpha antibody or a fragment thereof, an anti-IL23 antibody or a fragment thereof, an antibody that binds to a receptor of IL23 or a fragment thereof, an IL23 receptor inhibitor, and an immune checkpoint antibody such as an anti- PD-1 antibody .
  • the agent can also be a vaccine, such as a vaccine for preventing an infection selected from a group consisting of Vibrio, Cholera, Typhoid, Rotavirus, Tuberculosis, HIV, Flu, Ebola, and Sendai.
  • single domain antibodies e.g., VHH domains
  • a heterologous protein or polypeptide or fragment thereof, for example, to a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450 or about 500 amino acids, or over 500 amino acids
  • fusion proteins as well as uses thereof.
  • fusion proteins comprising an antigen-binding fragment of the single domain antibody provided herein (e.g., CDR1, CDR2, and/or CDR3) and a heterologous protein, polypeptide, or peptide.
  • an antibody that binds to a cell surface receptor expressed by a particular cell type may be fused or conjugated to a modified antibody provided herein.
  • antibodies provided herein can be fused to marker or “tag” sequences, such as a peptide, to facilitate purification.
  • the marker or tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (see, e.g., QIAGEN, Inc.), among others, many of which are commercially available.
  • a pQE vector see, e.g., QIAGEN, Inc.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and the “FLAG” tag.
  • HA hemagglutinin
  • FLAG FLAG
  • Fusion proteins may be generated, for example, through the techniques of gene- shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”).
  • DNA shuffling may be employed to alter the activities of the single domain antibodies as provided herein, including, for example, antibodies with higher affinities and lower dissociation rates (see, e.g., U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol.8:724-33; Harayama, 1998, Trends Biotechnol.16(2):76-82; Hansson et al., 1999, J. Mol. Biol.287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13).
  • Antibodies, or the encoded antibodies may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination.
  • a polynucleotide encoding an antibody provided herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • a single domain antibody provided herein e.g., VHH domain
  • a second antibody to form an antibody heteroconjugate as described, for example, in U.S. Pat. No.4,676,980.
  • Antibodies that bind to pIgR as provided herein may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • exemplary agents include, but are not limited to, a small molecule, a polynucleotide, a radioisotope, a toxin, an enzyme, an anticoagulant, a hormone, a cytokine, an anti-inflammatory molecule, an RNAi, a mRNA, a self-replicating RNA, an antibiotic, or an antibody-antibiotic conjugate.
  • the agent is an antibiotic.
  • antibiotics include, but are not limited to, macrolide antibiotic, a fluoroquinolone, a tetracycline, amoxicillin, ceftriaxone, penicillin G, linezolid, moxifloxacin, and azithromycin.
  • radioisotopes include, but are not limited to, from 18 F, 99 Tc, 111 In, 123 I, 201 Tl, 133 Xe, 11 C, 13 N, 15 O, 18 F, 62 Cu, 64 Cu, 124 I, 76 Br, 82 Rb, 89 Zr and 68 Ga.
  • antibodies provided herein are conjugated or recombinantly fused, e.g., to a diagnostic molecule.
  • Such diagnosis and detection can be accomplished, for example, by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin or avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, or aequorin; chemiluminescent material, such as, 225Acg-emitting,
  • radioactive isotopes include 3H, 11C, 13C, 15N, 18F, 19F, 55Co, 57Co, 60Co, 61Cu, 62Cu, 64Cu, 67Cu, 68Ga, 72As, 75Br, 86Y, 89Zr, 90Sr, 94mTc, 99mTc, 115In, 1231, 1241, 125I, 1311, 211At, 212Bi, 213Bi, 223Ra, 226Ra, 225Ac and 227Ac.
  • the linker may be a “cleavable linker” facilitating release of the conjugated agent in the cell, but non-cleavable linkers are also contemplated herein.
  • Linkers for use in the conjugates of the present disclosure include, without limitation, acid labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids, for example, valine and/or citrulline such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., Chari et al., 1992, Cancer Res.52:127-31; and U.S. Pat.
  • acid labile linkers e.g., hydrazone linkers
  • disulfide-containing linkers e.g., disulfide-containing linkers
  • peptidase-sensitive linkers e.g., peptide linkers comprising amino acids, for example, valine and/or citrulline such as citrulline-valine or phenylalanine-lysine
  • photolabile linkers
  • Conjugates of the antibody and agent may be made using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo- MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4- vinylsulfone)benzoate).
  • bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo
  • conjugates of antibodies and agents may be prepared using any suitable methods as disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed.2008)).
  • Conventional conjugation strategies for antibodies and agents have been based on random conjugation chemistries involving the e-amino group of Lys residues or the thiol group of Cys residues, which results in heterogenous conjugates.
  • Recently developed techniques allow site-specific conjugation to antibodies, resulting in homogeneous loading and avoiding conjugate subpopulations with altered antigen-binding or pharmacokinetics.
  • thiomabs comprising cysteine substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth.332: 41-52; and Junutula et al., 2008, Nature Biotechnol.26:925-32).
  • selenocysteine is cotranslationally inserted into an antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56; and Hofer et al., 2009, Biochemistry 48(50):12047-57). 5.3.1. Methods of Making a Genetically Fused Protein [00343]
  • the single domain antibody is genetically fused to the agent.
  • the linker may be a flexible linker comprising a sequence selected from the group consisting of EPKTPKPQPQPQLQPQPNPTTESKSPK (SEQ ID NO: 130), (EAAAK)n (SEQ ID NO: 147), (GGGGS)n (SEQ ID NO: 148) and (GGGS)n (SEQ ID NO: 149), wherein n is an integer from 1 to 20.
  • the single domain antibody is genetically conjugated to a therapeutic molecule, with a hinge region linking the single domain antibody to the therapeutic molecule.
  • the hinge region may be a flexible linker comprising a sequence selected from the group consisting of EPKTPKPQPQPQLQPQPNPTTESKSPK (SEQ ID NO: 130), (EAAAK)n (SEQ ID NO: 147), (GGGGS)n (SEQ ID NO: 148) and (GGGS)n (SEQ ID NO: 149), wherein n is an integer from 1 to 20.
  • the hinge region comprises the sequence EPKTPKPQPQPQLQPQPNPTTESKSPK (SEQ ID NO: 130), or comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with EPKTPKPQPQPQLQPQPNPTTESKSPK (SEQ ID NO: 130).
  • the hinge region comprises the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 150), or comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with EPKSCDKTHTCPPCP (SEQ ID NO: 150).
  • the hinge region comprises the sequence ERKCCVECPPCP (SEQ ID NO: 151), or comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with ERKCCVECPPCP (SEQ ID NO: 151).
  • the hinge region comprises the sequence ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP) 3 (SEQ ID NO: 152), or comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP) 3 (SEQ ID NO: 152).
  • the hinge region comprises the sequence ESKYGPPCPSCP (SEQ ID NO: 153), or comprises an amino acid sequence having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with ESKYGPPCPSCP (SEQ ID NO: 153).
  • ESKYGPPCPSCP SEQ ID NO: 153
  • the fusion protein provided herein is recombinantly expressed.
  • Recombinant expression of a fusion protein provided herein may require construction of an expression vector containing a polynucleotide that encodes the protein or a fragment thereof.
  • the vector for the production of the molecule may be produced by recombinant DNA technology using techniques well-known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control signals.
  • replicable vectors comprising a nucleotide sequence encoding a fusion protein provided herein, or a fragment thereof, or a CDR, operably linked to a promoter.
  • the expression vector can be transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce a fusion protein provided herein.
  • host cells containing a polynucleotide encoding a fusion protein provided herein or fragments thereof operably linked to a heterologous promoter are also provided herein.
  • a variety of host-expression vector systems may be utilized to express the fusion protein provided herein (see, e.g., U.S. Patent No.5,807,715).
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express a fusion protein provided herein in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
  • Bacterial cells such as Escherichia coli, or, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, can be used for the expression of a recombinant fusion protein.
  • mammalian cells such as Chinese hamster ovary cells (CHO)
  • CHO Chinese hamster ovary cells
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies or variants thereof (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2).
  • fusion proteins provided herein are produced in CHO cells.
  • the expression of nucleotide sequences encoding the fusion proteins provided herein is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the fusion protein being expressed. For example, when a large quantity of such a fusion protein is to be produced, for the generation of pharmaceutical compositions of a fusion protein, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E.
  • coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.24:5503-5509); and the like.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • an AcNPV promoter for example the polyhedrin promoter.
  • the coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing the fusion protein in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359).
  • Specific initiation signals may also be required for efficient translation of inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.
  • cell lines which stably express the fusion proteins may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the fusion protein.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the binding molecule.
  • a number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci.
  • adenine phosphoribosyltransferase genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O’Hare et al., 1981, Proc. Natl. Acad. Sci.
  • the host cell may be co-transfected with multiple expression vectors provided herein.
  • the vectors may contain identical selectable markers which enable equal expression of respective encoding polypeptides.
  • a single vector may be used which encodes, and is capable of expressing multiple polypeptides.
  • the coding sequences may comprise cDNA or genomic DNA.
  • a fusion protein provided herein may be purified by any method known in the art for purification of a polypeptide (e.g., an immunoglobulin molecule), for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, sizing column chromatography, and Kappa select affinity chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, sizing column chromatography, and Kappa select affinity chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the fusion protein molecules provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. 5.4.
  • the disclosure provides polynucleotides that encode the single domain antibodies that bind to pIgR and fusion proteins comprising the single domain antibodies that bind ot pIgR described herein.
  • the polynucleotides of the disclosure can be in the form of RNA or in the form of DNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non- coding (anti-sense) strand.
  • the polynucleotide is in the form of cDNA.
  • the polynucleotide is a synthetic polynucleotide.
  • the nucleic acid molecule provided herein comprises a sequence that encodes the single domain antibody having the sequence of: QVQLVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGSIDLNWYGGMD YWGQGTQVTVSS (SEQ ID NO: 93), EVQVVESGGGLVQAGGSLKLACAAPGLTFSSYRMGWFRQAPGQEREFVAAIDWNGRG TYYRYYADSVKGRSTISRDNAKNTVYLQMNSLKPEDTAVYYCAATTVLTDPRVLNEYA TWGQGTQVTVSS (SEQ ID NO: 94), QLQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWY
  • the nucleic acid molecule comprises the sequence of: CAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAA ACTCGCCTGTGCAGCACCTGGACTTACCTTCAGTTCGTATCGCATGGGCTGGTTCCG CCAGGCTCCAGGGCAGGAGCGTGAGTTTGTAGCAGCTATTGATTGGAATGGTCGTG GCACATATTATCGATACTATGCAGACTCCGTGAAGGGCCGATCCACCATTTCCAGAG ACAACGCCAAGAACACGATGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACG GCCGTTTATTACTGTGCAGCAGGTTCGATCGACCTTAACTGGTACGGCGGCATGGAC TACTGGGGCNANGGGACCCAGGTCACCGTCCTCA (SEQ ID NO: 133), GAGGTGCAGGTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAA ACTCGCCTGTGCAGCACCTGGACTTACC
  • nucleic acid molecules described herein can be incorporated into a recombinant expression vector.
  • the present disclosure provides recombinant expression vectors comprising any of the nucleic acids of the disclosure.
  • the term “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
  • the vectors described herein are not naturally-occurring as a whole; however, parts of the vectors can be naturally-occurring.
  • the described recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturally-occurring or non-naturally-occurring internucleotide linkages, or both types of linkages. The non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
  • the recombinant expression vector of the disclosure can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host.
  • Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.).
  • Bacteriophage vectors such as lGT10, lGT11, lEMBL4, and lNM1149, lZapII (Stratagene) can be used.
  • plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech).
  • the recombinant expression vector may be a viral vector, e.g., a retroviral vector, e.g., a gamma retroviral vector.
  • the recombinant expression vectors are prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • Constructs of expression vectors which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.
  • Replication systems can be derived, e.g., from ColE1, SV40, 2m plasmid, l, bovine papilloma virus, and the like.
  • the recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, plant, fungus, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based.
  • the recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.
  • Suitable marker genes for the described expression vectors include, for instance, neomycin/G418 resistance genes, histidinol x resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • the recombinant expression vector can comprise a native or normative promoter operably linked to the nucleotide sequence of the disclosure.
  • the selection of promoters e.g., strong, weak, tissue-specific , inducible and developmental-specific, is within the ordinary skill of the artisan.
  • the combining of a nucleotide sequence with a promoter is also within the skill of the artisan.
  • the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an RSV promoter, an SV40 promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.
  • CMV cytomegalovirus
  • RSV cytomegalovirus
  • SV40 SV40 promoter
  • the recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression. [00368] Further, the recombinant expression vectors can be made to include a suicide gene.
  • suicide gene refers to a gene that causes the cell expressing the suicide gene to die.
  • the suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent.
  • agent genes include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.
  • the present disclosure further relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and/or derivatives of the single domain antibody that binds pIgR of the disclosure.
  • the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding the single domain antibody that binds pIgR of the disclosure.
  • a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence” is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence.
  • These mutations of the reference sequence can occur at the 5 ⁇ or 3 ⁇ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both.
  • a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide.
  • a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code).
  • Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.
  • a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide.
  • a polynucleotide variant is produced to increase expression of the encoded polypeptide.
  • a polynucleotide variant is produced to decrease expression of the encoded polypeptide.
  • a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • a polynucleotide is isolated.
  • a polynucleotide is substantially pure.
  • host cells comprising the nucleic acid molecules described herein.
  • the host cell may be any cell that contains a heterologous nucleic acid.
  • the heterologous nucleic acid can be a vector (e.g., an expression vector).
  • a host cell can be a cell from any organism that is selected, modified, transformed, grown, used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme.
  • An appropriate host may be determined.
  • the host cell may be selected based on the vector backbone and the desired result.
  • a plasmid or cosmid can be introduced into a prokaryote host cell for replication of several types of vectors.
  • Bacterial cells such as, but not limited to DH5a, JM109, and KCB, SURE® Competent Cells, and SOLOPACK Gold Cells, can be used as host cells for vector replication and/or expression. Additionally, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to yeast (e.g., YPH499, YPH500 and YPH501), insects and mammals.
  • yeast e.g., YPH499, YPH500 and YPH501
  • mammalian eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, Saos, PC12, SP2/0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No.85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines.
  • An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196).
  • compositions comprising a single domain antibody or a therapeutic molecule of the present disclosure.
  • a pharmaceutical composition comprises therapeutically effective amount of the antibody or therapeutic molecule provided herein and a pharmaceutically acceptable excipient.
  • excipient can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.
  • Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is an exemplary excipient. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.
  • Such compositions will contain a prophylactically or therapeutically effective amount of the antibodies or therapeutic molecules provided herein, such as in purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the single domain antibody or therapeutic molecule provided herein may be formulated in any suitable form for delivery to a target cell/tissue, e.g., as microcapsules or macroemulsions (Remington, supra; Park et al., 2005, Molecules 10:146-61; Malik et al., 2007, Curr. Drug. Deliv.4:141-51), as sustained release formulations (Putney and Burke, 1998, Nature Biotechnol. 16:153-57), or in liposomes (Maclean et al., 1997, Int. J. Oncol.11:325-32; Kontermann, 2006, Curr. Opin. Mol. Ther.8:39-45).
  • a target cell/tissue e.g., as microcapsules or macroemulsions (Remington, supra; Park et al., 2005, Molecules 10:146-61; Malik et al., 2007, Curr. Drug. Deliv.4:141
  • the single domain antibody or therapeutic molecule provided herein can also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly- (methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • compositions and delivery systems are known and can be used with the single domain antibody or therapeutic molecule provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the single domain antibody or therapeutic molecule provided herein, construction of a nucleic acid as part of a retroviral or other vector, etc.
  • the antibody or therapeutic molecule provide herein is formulated in a pharmaceutic composition suitable for less-invasive or non-invasive administration.
  • the antibody or therapeutic molecule provide herein is formulated in a pharmaceutic composition suitable for oral administration.
  • the antibody or therapeutic molecule provide herein is formulated in a pharmaceutic composition suitable for buccal administration. In a specific embodiment, the antibody or therapeutic molecule provide herein is formulated in a pharmaceutic composition suitable for inhalation administration. In a specific embodiment, the antibody or therapeutic molecule provide herein is formulated in a pharmaceutic composition suitable for nasal administration.
  • Non-limiting exemplary dosage forms are described in more detail in the following sections. 5.5.1. Oral Dosage Forms [00381] In certain embodiments, the antibodies or therapeutic molecules provided herein are formulated in pharmaceutical compositions suitable for oral administration.
  • Oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups).
  • dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).
  • Typical oral dosage forms are prepared by combining the active ingredients in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration.
  • excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.
  • excipients suitable for use in solid oral dosage forms include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.
  • tablets and capsules represent advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.
  • Such dosage forms can be prepared by any of the methods of pharmacy.
  • pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
  • a tablet can be prepared by compression or molding.
  • Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient.
  • Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • excipients that can be used in oral dosage forms provided herein include, but are not limited to, binders, fillers, disintegrants, and lubricants.
  • Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos.2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.
  • Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA), and mixtures thereof.
  • An specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581.
  • Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103TM and Starch 1500 LM.
  • fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
  • the binder or filler in pharmaceutical compositions provided herein is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.
  • Disintegrants are used in compositions to provide tablets that disintegrate when exposed to an aqueous environment.
  • Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions.
  • a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms.
  • the amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art.
  • Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, preferably from about 1 to about 5 weight percent of disintegrant.
  • Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
  • Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.
  • hydrogenated vegetable oil e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil
  • zinc stearate ethyl oleate, ethyl laureate, agar, and mixtures thereof.
  • Additional lubricants include, for example, a syloid silica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore, MD), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Plano, TX), CAB-O-SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, MA), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated. 5.5.2.
  • AEROSIL200 syloid silica gel
  • a coagulated aerosol of synthetic silica marketed by Degussa Co. of Plano, TX
  • CAB-O-SIL a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, MA
  • Topical and Mucosal dosage forms provided herein include, but are not limited to, sprays, aerosols, solutions, emulsions, suspensions, eye drops or other ophthalmic preparations, or other forms known to one of skill in the art. See, e.g., Remington’s Pharmaceutical Sciences, 16 th and 18 th eds., Mack Publishing, Easton PA (1980 & 1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985).
  • the mucosal dosage forms provided herein are suitable for administration to oral mucosal surface (buccal) or to nasal mucosal surface of a subject.
  • Suitable excipients e.g., carriers and diluents
  • excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form solutions, emulsions or gels, which are non-toxic and pharmaceutically acceptable.
  • Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington’s Pharmaceutical Sciences, 16 th and 18 th eds., Mack Publishing, Easton PA (1980 & 1990).
  • the pH of a pharmaceutical composition or dosage form may also be adjusted to improve delivery of one or more active ingredients.
  • the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery.
  • Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery.
  • stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery-enhancing or penetration-enhancing agent.
  • Different salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition. 5.5.3. Delayed Release Dosage Forms [00394]
  • a pharmaceutical composition can be provided as a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release (see, e.g., Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng.
  • polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., a fusion protein as described herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol.
  • a prophylactic or therapeutic agent e.g., a fusion protein as described herein
  • a composition provided herein see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol.
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol.2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, 1990, Science 249:1527-33. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents as described herein (see, e.g., U.S. Pat. No.4,526,938, PCT publication Nos.
  • the single domain antibodies are useful for transporting an agent from an apical surface of a pIgR- expressing cell to a basolateral surface of the pIgR-expressing cell, and can deliver the agent, e.g., to systemic circulation or lamina intestinal or gastrointestinal tract of a subject, via methods such as oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4.
  • the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5.
  • the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6.
  • the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7.
  • the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9.
  • the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10. In yet another specific embodiment, the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12.
  • a method for delivering from an apical surface of a pIgR-expressing cell to a basolateral surface of the pIgR-expressing cell comprising contacting the pIgR-expressing cell with (i) a single domain antibody that binds to pIgR provided herein, or (ii) a therapeutic molecule comprising an agent and the single domain antibody.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4.
  • the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5.
  • the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6.
  • the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7.
  • the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9.
  • the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10. In yet another specific embodiment, the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00398] In some embodiments, provide herein is a single domain antibody that binds to pIgR provided herein for use in delivering an agent from an apical surface of a pIgR-expressing cell to a basolateral surface of the pIgR-expressing cell, wherein the agent is conjugated to the single domain antibody.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4.
  • the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5.
  • the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6.
  • the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7. In yet another specific embodiment, the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10. In yet another specific embodiment, the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12.
  • a single domain antibody that binds to pIgR provided herein for delivering an agent from an apical surface of a pIgR-expressing cell to a basolateral surface of the pIgR-expressing cell, wherein the agent is conjugated to the single domain antibody.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7. In yet another specific embodiment, the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10.
  • the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00400] In other embodiments, provided herein is a method for transporting a therapeutic molecule to a basolateral surface of the pIgR-expressing cell of a subject, comprising administering to the subject the therapeutic molecule comprising an agent and a VHH domain. In some embodiments, the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery. In a specific embodiment, the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2. In another specific embodiment, the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3. In yet another specific embodiment, the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7.
  • the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10. In yet another specific embodiment, the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00401] In other embodiments, provided herein is a single domain antibody for use in transporting a therapeutic molecule to a basolateral surface of the pIgR-expressing cell of a subject, wherein the therapeutic molecule comprises an agent and the single domain antibody.
  • the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4.
  • the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5.
  • the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7. In yet another specific embodiment, the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10. In yet another specific embodiment, the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12.
  • a single domain antibody for transporting a therapeutic molecule to a basolateral surface of the pIgR-expressing cell of a subject, wherein the therapeutic molecule comprises an agent and the single domain antibody.
  • the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7. In yet another specific embodiment, the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10.
  • the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00403] In yet other embodiments, provided herein is a method for transporting a therapeutic molecule to systemic circulation of a subject, comprising administering to the subject the therapeutic molecule comprising an agent and a single domain antibody, wherein the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery. In a specific embodiment, the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2. In another specific embodiment, the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3. In yet another specific embodiment, the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7.
  • the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10. In yet another specific embodiment, the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12.
  • a single domain antibody for use in transporting a therapeutic molecule to systemic circulation of a subject, wherein the therapeutic molecule comprises the single domain antibody and an agent, and wherein the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7. In yet another specific embodiment, the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10.
  • the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00405] In yet other embodiments, provided herein is a use of VHH for transporting a therapeutic molecule to systemic circulation of a subject, wherein the therapeutic molecule comprises the single domain antibody and an agent, and wherein the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • a method for transporting a therapeutic molecule to lamina intestinal or gastrointestinal tract of a subject comprising administering to the subject the therapeutic molecule comprising an agent and a single domain antibody, wherein the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7. In yet another specific embodiment, the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10.
  • the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00407] In yet other embodiments, provided herein is a single domain antibody for use in transporting a therapeutic molecule to lamina intestinal or gastrointestinal tract of a subject, wherein the therapeutic molecule comprises an agent and the single domain antibody, and wherein the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery. In a specific embodiment, the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2. In another specific embodiment, the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3. In yet another specific embodiment, the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7.
  • the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10. In yet another specific embodiment, the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12.
  • a single domain antibody for transporting a therapeutic molecule to lamina intestinal or gastrointestinal tract of a subject, wherein the therapeutic molecule comprises an agent and the single domain antibody, and wherein the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7. In yet another specific embodiment, the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10.
  • the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00409] In some embodiments of the various methods and uses provided herein, the therapeutic agent is transported from an apical surface of a pIgR-expressing cell to a basolateral surface of the pIgR-expressing cell in the subject. [00410] In some embodiments, the single domain antibody or the therapeutic molecule comprising an agent and the single domain antibody is also capable of being transported from the basolateral surface of the pIgR-expressing cell to the apical surface of the pIgR-expressing cell.
  • a method of treating a disease or disorder comprising administering a therapeutic molecule comprising an agent and the single domain antibody provided herein to a subject, wherein optionally the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery.
  • the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7. In yet another specific embodiment, the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10.
  • the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00412] In yet other embodiments, provided herein is a therapeutic molecule comprising an agent and a single domain antibody provided herein for use in treating a disease or disorder in subject, wherein optionally the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery. In a specific embodiment, the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1. In another specific embodiment, the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2.
  • the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3.
  • the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4.
  • the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5.
  • the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6.
  • the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7.
  • the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9.
  • the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10. In yet another specific embodiment, the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00413] In yet other embodiments, provided herein is a use of a therapeutic molecule comprising an agent and a single domain antibody provided herein for treating a disease or disorder in subject, wherein optionally the therapeutic molecule is administered to the subject via oral delivery, buccal delivery, nasal delivery or inhalation delivery. In a specific embodiment, the single domain antibody is VHH1 or a VHH having the same CDRs as VHH1.
  • the single domain antibody is VHH2 or a VHH having the same CDRs as VHH2. In another specific embodiment, the single domain antibody is VHH3 or a VHH having the same CDRs as VHH3. In yet another specific embodiment, the single domain antibody is VHH4 or a VHH having the same CDRs as VHH4. In yet another specific embodiment, the single domain antibody is VHH5 or a VHH having the same CDRs as VHH5. In yet another specific embodiment, the single domain antibody is VHH6 or a VHH having the same CDRs as VHH6. In yet another specific embodiment, the single domain antibody is VHH7 or a VHH having the same CDRs as VHH7.
  • the single domain antibody is VHH9 or a VHH having the same CDRs as VHH9. In yet another specific embodiment, the single domain antibody is VHH10 or a VHH having the same CDRs as VHH10. In yet another specific embodiment, the single domain antibody is VHH11 or a VHH having the same CDRs as VHH11. In yet another specific embodiment, the single domain antibody is VHH12 or a VHH having the same CDRs as VHH12. [00414] In some embodiments, the disease or disorder is a metabolic disease or disorder. In some embodiments, the disease or disorder is diabetes. In some embodiments, the disease or disorder is cancer. In other embodiments, the disease or disorder is an immune disease or disorder.
  • the disease or disorder is a gastrointestinal disease. In some embodiments, the disease or disorder is gastrointestinal inflammation. In some embodiments, the disease or disorder is inflammatory bowel disease (IBD). In some embodiments, the disease or disorder is Crohn’s disease (CD). In some embodiments, the disease or disorder is ulcerative colitis (UC). In some embodiments, the disease or disorder is ankylosing spondylitis (AS). In some embodiments, the disease or disorder is colitis.
  • IBD inflammatory bowel disease
  • CD Crohn’s disease
  • UC ulcerative colitis
  • AS ankylosing spondylitis
  • the disease or disorder is colitis.
  • the single domain antibodies of the disclosure may be conjugated to any agent that can be used to treat or ameliorate symptoms of intestinal inflammation, IBD, UC or AS, including agents which are inhibitors of pro-inflammatory cytokines, inhibitors of Th17 cell activation and/or differentiation, molecules inhibiting lymphocyte trafficking or adhesion, modulators of innate immune system, modulators of macrophages, dendritic cells, regulatory T cells (Treg) or effector CD8 + or CD4 + T cells.
  • agents which are inhibitors of pro-inflammatory cytokines, inhibitors of Th17 cell activation and/or differentiation, molecules inhibiting lymphocyte trafficking or adhesion, modulators of innate immune system, modulators of macrophages, dendritic cells, regulatory T cells (Treg) or effector CD8 + or CD4 + T cells.
  • Such exemplary agents include inhibitors of TNF-a IL-12, IL-6, IL-13, IL-17A, IL17A/F, IL-18, IL-21, modulators of TLR3 or TLR4 pathway, TNF- ⁇ inhibitors infliximab, adalimumab, certolizumab, golimumab, etanercept and biosimilars thereof, IL-23 inhibitors ustekinumab, risankizumab, brazikumab and mirikizumab, IL-23 receptor inhibitors, IL-17 inhibitor secukinumab, IL-6 inhibitors tocilizumab and PF- 04236921, PDE4 inhibitor apermilast, JAK inhibitors tocacifinib, filgotinib, upadacitinib or peficiting, inhibitors of cell adhesion such as natalizumab, vedolizumab, etrolizumab, abrilumab,
  • the agent is an inhibitor of IL-23 receptor.
  • the agent targeting pathogenic pathways in intestinal inflammation herein may be a known molecule, a variant or a fragment of the known molecule, or generated de novo and genetically fused or chemically conjugated to the single domain antibody of the disclosure using known methods and those described herein.
  • the methods or uses provided here are for delivering a vaccine for preventing an infection, such as Vibrio, Cholera, Typhoid, Rotavirus, Tuberculosis, HIV, Flu, Ebola, and Sendai.
  • the agent in the therapeutic molecule comprises a peptide.
  • the agent in the therapeutic molecule comprises an antibody or a fragment thereof. In some embodiments of the various methods and uses provided herein, the agent in the therapeutic molecule comprises a peptide conjugated to a small molecule compound (e.g., antibody drug conjugate). In some embodiments of the various methods and uses provided herein, the agent in the therapeutic molecule comprises a nucleic acid. In some embodiments of the various methods and uses provided herein, the agent in the therapeutic molecule comprises a vaccine.
  • a prophylactic or therapeutic agent e.g., an antibody or therapeutic molecule
  • a composition provided herein that will be effective in the prevention and/or treatment of a disease or condition
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and should be decided according to the judgment of the practitioner and each patient’s circumstances.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the route of administration for a dose of an antibody or therapeutic molecule provided herein to a patient is oral delivery, buccal delivery, nasal delivery, inhalation delivery, or a combination thereof, but other routes may be also acceptable.
  • Each dose may or may not be administered by an identical route of administration.
  • an antibody or therapeutic molecule provided herein may be administered via multiple routes of administration simultaneously or subsequently to other doses of the same or a different agent provided herein. [00420]
  • certain abbreviations are used herein.
  • One example is the single letter abbreviation to represent amino acid residues.
  • amino acids and their corresponding three letter and single letter abbreviations are as follows: alanine Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acid Asp (D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (Q) glycine Gly (G) histidine His (H) isoleucine Ile (I) leucine Leu (L) lysine Lys (K) methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser (S) threonine Thr (T) tryptophan Trp (W) tyrosine Tyr (Y) valine Val (V) [00421] The disclosure is generally disclosed herein using affirmative language to describe the numerous embodiments.
  • Example 1 Immunization, recovery and screening of pIgR binders
  • llamas were immunized with recombinant human pIgR (hpIgR) and/or mouse pIgR (mpIgR) for about 90 days.
  • the whole blood and PBMCs was isolated from llamas, and RNA was prepared.
  • llama-specific primers annealing to (i) the VH (heavy-chain variable region), (ii) VHH leader sequence genes, and (iii) the CH2 gene were used to PCR amplify the VH and VHH gene repertoires.
  • VHH repertoires were separated from VH repertoires by running the PCR fragments on a gel and excising the smaller band.
  • the VHH gene repertoire was reamplified and cloned into a CMV-based mammalian vector.
  • the VHH-gene was formatted as Ig-fusion.
  • the library was transformed in E. coli. Single colonies were picked in a 96-well format for Sanger sequencing. From approximately 300 unique sequences, a select number of VHH sequences were selected for miniprep DNA, and then scaled-up for future recombinant expression and screening.
  • the sequence of the human IgG1 mono-Fc protein is as follows: SPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVH NA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKG QPREPQ VYTKPPSREE MTKNQVSLSC LVKGFYPSDI AVEWESNGQP ENNYKTTVPV L DSDGSFRLA SYLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 146) [00427] This VHH panel was screened for binding to hpIgR and mpIgR ectodomain by enzyme- linked immunosorbent assays (ELISAs) resulted in 40 positive hits.
  • ELISAs enzyme- linked immunosorbent assays
  • Bio-layer Interferometry was performed as follows.
  • the ForteBioOctet RED384 system (Pall Corporation) was used to measure binding kinetics between VHH-mono-Fc molecules and pIgR proteins, and between IgA and pIgR proteins (in the absence and presence of VHH-mono-Fc molecules).
  • Data were collected with Octet Data Acquisition version 7.1.0.87 (ForteBio) and analyzed using Octet Data Analysis version 7.1 (ForteBio).
  • VHH-mono-Fc was immobilized on amine-reactive generation-2 (ARG2) biosensors according to manufacturer’s instructions and increasing concentrations of pIgR proteins were exposed to sensor-immobilized VHH.
  • ARG2 amine-reactive generation-2
  • HIS-tagged pIgR proteins were immobilized on anti-HIS biosensors and exposed to increasing concentrations of VHH-mono-Fc molecules.
  • Association and dissociation rates were measured by the shift in wavelength (nm). For each sensor-immobilized protein, at least three different ligand concentrations were used, and K D (equilibrium dissociation constant) was obtained by fitting the data to 1:1 binding model.
  • DNA constructs for VHH were sub-cloned into mammalian expression vectors using the In-Fusion ® HD Cloning Kit.
  • ExpiCHO TM cells were transfected with the appropriate expression vectors.
  • Supernatants were harvested after 6-7 days by centrifugation (4,000 g, 15 min), passed through a 0.45-um filter, and purified at 4 o C by MabSelect TM SuRe TM chromatography on an ⁇ KTA express system (both GE Healthcare) using DPBS (Sigma) as running buffer and 0.1 M sodium acetate, pH 3.5 as elution buffer.
  • HEK Expi293 TM cells were transfected with pIgR-domain expression vectors using ExpiFectamine TM 293 transfection kit. Supernatants were harvested after 6-7 days by centrifugation (4,000 g, 15 min), passed through a 0.45-um filter and purified by immobilized metal ion chromatography using HisPur TM Cobalt resin (Thermo scientific). Buffer NPI-20 (Teknova) was used as running buffer and Buffer NPI-300 (Teknova) containing 300mM Imidazole was used as elution buffer.
  • the 10 pIgR binders were expressed and purified from CHO cells using Protein-A affinity chromatography. Size-exclusion chromatography combined with multi-angle light scattering showed that molecular weight of 10 VHH-mono-Fc binders (VHH2, VHH3, VHH4, VHH5, VHH6, VHH7, VHH9, VHH10, VHH11, and VHH12) ranged from 41.3 kDa to 48.7 kDa.
  • Thermal stability of a sample was determined by differential scanning fluorimetry, specifically the NanoDSF method, using an automated Prometheus instrument. Measurements were made by loading a sample into a 24-well capillary from a 384-well sample plate. Duplicate runs were performed for each sample.
  • a Prometheus NanoDSF user interface (Melting Scan tab) was used to set up the experimental parameters for the run.
  • the thermal scans for a typical IgG sample spanned from 20 ⁇ C to 95 ⁇ C at a rate of 1.0 ⁇ C/minute.
  • Dual-UV technology monitoring of intrinsic tryptophan and tyrosine fluorescence at the emission wavelengths of 330 nm and 350 nm was undertaken.
  • the F350 nm/F330 nm ratio was plotted against temperature to generate an unfolding curve.
  • the back reflection optics of the instrument was also used for the detection of sample aggregation. Such optics emitted near-UV light at a wavelength that is not absorbed by proteins.
  • VHH-mono-Fc molecules were expressed in CHO cells and purified using Protein-A affinity chromatography.
  • VHH-mono-Fc molecules recognize cell-surface hpIgR
  • MDCK cells Madin-Darby canine kidney (MDCK) cells, a commonly used epithelia model system, were used to investigate if VHH binders could be transported across epithelia by pIgR mediated transcytosis.
  • MDCK cells un-transfected or stably transfected with human pIgR were used to study transcytosis (See Natvig, I.B., Johansen, F.E., Nordeng, T.W., Haraldsen, G. & Brandtzaeg, P. Mechanism for enhanced external transfer of dimeric IgA over pentameric IgM: studies of diffusion, binding to the human polymeric Ig receptor, and epithelial transcytosis. J. Immunol.159, 4330-4340 (1997)).
  • hpIgR hpIgR in MDCK cells and monolayer formation were confirmed by confocal laser microscopy.
  • VHH- mono-Fc that did not bind to pIgR was used as a control together with 100 nM (15mg/mL) human IgG (to control for unspecific transport and leakage).
  • the apical medium was harvested, and the amount of VHHmono-Fc, transported by pIgR, was calculated by standard titration studies. IgG leakage to the apical medium was detected by MSD. The results of the transcytosis assay are shown in Figures 12A-12B.
  • a biotinylated anti-VHH antibody was used to capture VHH-mono-Fc on streptavidin plates and a ruthenylated anti-Fc antibody to detect VHH-mono-Fc by the MSD platform.
  • the results of this assay are shown in Figure 12C.
  • Six VHHs (2, 4, 6, 9, 11 and 12) showed >10-fold increase in their apical concentration relative to control VHH. 6.4.
  • Example 4 Transcytosis assays using primary human lung tissue model [00444] The EpiAirway human lung tissue model was also used to test the transcytosis activity of 10 VHH molecules from the basolateral to the apical epithelium and their delivery to the mucosal lumen.
  • the EpiAirway model is depicted in Figure 18.
  • the EpiAirway model is an established lung tissue model engineered from primary human tracheal bronchial cells. Tissue models were obtained from Mattek Corporation and maintained according to manufacturer’s instructions.20 mg of test and control VHH-mono-Fc molecules were added to 1 ml of EpiAirway media in the basolateral chamber and 100 ul of samples were collected from the basolateral and apical chambers at 0, 24 and 48 hours. EpiAirway TEER buffer was used to collect the mucus from the apical chambers. The amount of VHH-mono-Fc present in basolateral media and apical mucus was quantified by electrochemiluminescence method.
  • streptavidin MSD plates were coated with a biotinylated anti-VHH antibody (2 mg/ml in PBS) for 1 hour at RT with 1000 rpm, washed 3X with PBT, incubated with blocking buffer for 1 hour at RT, incubated with VHH-mono-Fc containing media/mucus (at different dilutions) for 2 hours at RT with 1000 rpm, washed 3X with PBT, incubated with ruthenylated-anti-human-Fc antibody (2 mg/ml in PBS) for 1 hour at RT with 1000 rpm, washed 3X with PBT and read plates in 40 ul reading buffer using the MSD imager.
  • VHH-mono-Fc The amount of VHH-mono-Fc in basolateral and apical chambers was calculated by plotting ECLU values against VHH-mono-Fc standard curves in Prism (Graphpad). The data is shown in Figure 19. A similar experiment in which IgG and IgA were transcytosed is shown in Figure 20. Each photomicrograph in Figure 20 is a representative image of one of the squares in the heat map in Figure 5. [00445] Figure 22 shows 3D reconstruction shows localization of hpIgR and VHH to the apical surface of the EpiAirway model. [00446] The amount of VHH present in the apical mucus 0, 24 and 48 hours post treatment was quantified by the electrochemiluminescence.
  • the Electrochemiluminescence assay was performed as follows. A meso-scale discovery (MSD) platform was used for conducting epitope mapping and epitope burial studies. To test the binding of VHH-mono-Fc molecules to purified pIgR protein constructs, Streptavidin MSD plates were coated with a biotinylated anti-HIS antibody (2 mg/ml in PBS) for 1 hour at RT with 1000 rpm, washed 3X with PBT (PBS + 0.1% Tween-20), incubated with blocking buffer for 1 hour at RT, incubated with His-tagged pIgR proteins (10 mg/ml in PBS) for 2 hours at RT with 1000 rpm, washed 3X with PBT, incubated with VHH-mono-Fc molecules (100 mg/ml in PBS) for 2 hours at RT with 1000 rpm, washed 3X with PBT, incubated with ruthenylated-anti- human-Fc antibody (2 mg
  • ECLU values were plotted as a heatmap. [00448] To check whether VHH recognizes a buried epitope on pIgR, EC 50 values were measured for VHH-mono-Fc molecules binding to hpIgR-ECD protein by electrochemiluminescence using two different detection antibodies, an anti-Fc antibody and an anti-VHH antibody.
  • pIgR ECD (10 mg/ml in PBS) was coated on high-bind MSD pwlates for 2 hours at RT with 1000 rpm, incubated with blocking buffer for 1 hour at RT, incubated with VHH-mono-Fc molecules (increasing concentrations in PBS) for 2 hours at RT with 1000 rpm, washed 3X with PBT, incubated with ruthenylated secondary antibody (2 mg/ml in PBS) for 1 hour at RT with 1000 rpm, washed 3X with PBT and read plates in 40 ul reading buffer using the MSD imager.
  • EC50 was calculated by fitting log VHH concentration versus log ECLU in Prism (Graphpad).
  • Figure 23 shows that the EpiAirway tissue model is on a slanted membrane, which is not ideal for image analysis.
  • Figure 24 illustrates a strategy for Opera Phenix imaging and analysis to overcome slanted tissue issues with EpiAirway tissue model.
  • indirect immunofluorescence was used to trace the location and amount of hpIgR and VHH across the EpiAirway tissue model by Opera Phenix confocal laser microscopy.
  • Indirect immunofluorescence was used to track the amount of pIgR and VHH- mono-Fc retained across the EpiAirway model two-days post-treatment.
  • Tissue samples were rinsed in PBS, tissues were fixed with 2 ml of 10% Formalin at RT for 20 minutes, washed three times with 2 ml PBST (1% Triton-X100 in PBS) at RT for 10 minutes each (with gentle agitation), incubated with primary antibodies (500ul apical, 500ul basolateral) diluted in PBTG (PBST with 10% goat serum) for 2 hours at RT (with gentle agitation), washed two times with 2 ml PBTG at RT for 10 minutes each (with gentle agitation), incubated with secondary antibodies (100ul apical, 100ul basolateral) diluted in PBTG for 1 hour at RT (with gentle agitation) and washed two times with 2 ml PBTG at RT for 10 minutes each (with gentle agitation).
  • primary antibodies 500ul apical, 500ul basolateral
  • PBTG PBST with 10% goat serum
  • secondary antibodies 100ul apical, 100ul basolateral
  • the primary antibody mix contained mouse antibody and biotinylated anti IgA antibody both at 5 mg/ml.
  • the secondary antibody mix contained Alexa-Flour 488-labelled anti-mouse antibody (1:100 dilution), Alexa-Flour 647-labelled streptavidin (1:100 dilution) and Hoechst (1: 1000 dilution).
  • Fixed, permeabilized and stained tissues were imaged at 20X resolution (30-40 planes, 2 um distance) using Opera Phenix confocal laser microscopy. Image analysis was performed using the Harmony suite, fluorescence readouts were corrected for membrane auto-fluorescence, normalized for number of cells and plotted as heat maps in Prism (Graphpad). [00451] The data is shown in Figure 5.
  • Example 5 Domain-level epitope mapping [00453] To conduct domain-level epitope mapping of VHHs, seven HIS-tagged hpIgR constructs (D1, D2, D3, D5, D1-D2, D2-D3 and D4-D5) were expressed and purified each encoding one or two domains of hpIgR ECD from HEK293 cells using immobilized metal ion affinity chromatography. Two constructs, D4 and D3-D4, showed poor expression and purification and were not used for epitope mapping assays. Binding of VHH-mFc molecules were tested to immobilized pIgR constructs by the electrochemiluminescence method. Results from the binding assay are shown as a heat map in Figure 2.
  • VHH3 recognizes a complex epitope on the hpIgR domain-1 interface, and in particular, while no differences in EC 50 were observed for VHH2 (4 nM for both detection antibodies), VHH3 showed a 54-fold increase in EC50 due to anti-VHH detection. Together these experiments indicated that VHH2 and VHH3 recognize domain-1 in a different fashion, that could have attributed to their differences in function. [00455] Epitope mapping showed that VHH2, VHH6 and VHH12 binds hpIgR domain 1, 2 and 5, respectively, whereas VHH9 and VHH11 binds to hpIgR domains 4-5.
  • VHH-mono-Fc molecules The binding of VHH-mono-Fc molecules to immobilized pIgR constructs is summarized as a heat map in Figure 2.
  • the epitopes of VHH2 and VHH3 are primarily contained within hpIgR domain 1 (D1), and the epitopes of VHH4 and VHH6 are primarily contained within hpIgR domain 2 (D2).
  • D1 is necessary for IgA binding to hpIgR.
  • the epitopes of other six VHH molecules are primarily contained within hpIgR domains 4-5 (D4-D5).
  • KD values were measured for full-length hpIgR ECD binding to immobilized VHH-mono-Fc molecules in the absence and presence of dIgA2 by bio-layer interferometry (Figure 3A).
  • VHHs showed a 1.3 to 3.3-fold decrease in affinity for binding to hpIgR ECD due to the presence of dIgA.
  • Pre-bound IgA had a small negative effect on binding of VHHs to pIgR, possibly due to steric hindrance arising from bound dIgA or conformational rearrangement of hpIgR ECD.
  • K D values for a recombinant dimeric IgA2 construct binding to the hpIgR ectodomain were measured with and without the presence of VHH-mono-Fc molecules.
  • Example 6 VHH/IgA competition studies (binding and transcytosis) [00459] The differences in binding between VHH2, the transcytosis-positive domain-1 binder described above, and VHH3, a transcytosis-negative domain-1 binder, were compared. VHH3 binds stronger than VHH2. To test the importance of hpIgR domain-1 CDRs on VHH2 and VHH3 binding, each domain-1 CDR of human pIgR was swapped with the respective domain-1 CDR of teleost fish pIgR to make three new CDR-swapped hpIgR domain-1 constructs for use in binding studies.
  • hpIgR ECD Full-length hpIgR ECD was purchased from R&D Systems.
  • the five constructs (D1-D2, D1, D1_tCDR1, D1_tCDR2, D1_tCDR3) were expressed and purified from HEK293 cells using immobilized metal ion affinity chromatography.
  • Three hpIgR domain-1 CDR mutants (D1_tCDR1, D1_tCDR2, D1_tCDR3) contain respective teleost fish CDR on a hpIgR domain-1 framework. His-tagged pIgR constructs were immobilized on anti-HIS biosensors and binding of VHH-mono-Fc molecules to pIgR constructs were measured by bio- layer interferometry.
  • VHH2 and VHH3 The KD values for two VHH-mono-Fc molecules (VHH2 and VHH3) binding to six HIS-tagged pIgR constructs.
  • VHH2 and VHH3 showed similar binding profiles towards CDR2 and CDR3 of hpIgR domain-1, while having different binding profiles towards CDR1 of hpIgR domain-1.
  • the properties of VHH2 and VHH3 are summarized in Figure 31.
  • the data of Figure 34 show that VHH2 and VHH3 compete with each other for binding to hpIgR.
  • VHH2 and VHH3 overlap partial epitopes on domain-1 and thus competed with one another for binding to hpIgR.
  • binding assays suggested that VHH3 binds to a more hidden epitope on domain-1 relative to VHH2 (Table 2).
  • VHH3-treated EpiAirway tissue model retained more pIgR in the basolateral epithelium relative to VHH2 or no VHH ( Figure 5). Given that the domain-1 plays a crucial interface and role in the inactive to active transitioning of hpIgR, these results suggest that VHH3 binding could shift the pIgR equilibrium towards an inactive conformation.
  • FIG 25 shows structure of pIgR:IgA complex by constrained scattering modeling.
  • Example 7 Additional Transcytosis Assays
  • MDCK cells expressing hpIgR as described in Example 3 are a relevant epithelia model system and were used to assay forward and reverse transcytosis activities of VHH-mono- Fc molecules.
  • MDCK cells expressing hpIgR were cultured in DMEM containing 10% FBS at 37 °C with 5% CO2.
  • VHH-mono-Fc molecules were added to the basolateral chamber and 100 ml of media was collected from the basolateral and apical chambers at different time points following the addition of VHH-mono-Fc molecules (0, 4, 8, 12, 24, 36 and 48 hours).
  • VHH-mono-Fc molecules were added to the apical chamber and 100 ml of media was collected from the basolateral and apical chambers at different time points following the addition of VHH-mono-Fc (0, 4, 8, 12, 24, 36 and 48 hours).
  • the amount of VHH-mono-Fc present in basolateral and apical media was quantified by electrochemiluminescence method.
  • Streptavidin MSD plates were coated with a biotinylated anti-VHH antibody (2 mg/ml in PBS) for 1 hour at RT with 1000 rpm, washed 3X with PBT, incubated with blocking buffer for 1 hour at RT, incubated with VHH-mono-Fc containing media/mucus (at different dilutions) for 1 hour at RT with 1000 rpm, washed 3X with PBT, incubated with ruthenylated-anti-human-Fc antibody (2 mg/ml in PBS) for 1 hour at RT with 1000 rpm, washed 3X with PBT and read plates in 40 ml reading buffer using the MSD imager.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Diabetes (AREA)
  • Nutrition Science (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Pulmonology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)

Abstract

L'invention concerne un procédé d'administration d'un anticorps à domaine unique ou d'une molécule thérapeutique à partir d'une surface apicale d'une cellule exprimant un récepteur d'immunoglobuline polymère (pIgR) à une surface basolatérale de la cellule exprimant le plgR, comprenant la mise en contact de la cellule exprimant le plgR avec l'anticorps à domaine unique ou la molécule thérapeutique, l'anticorps à domaine unique se liant au plgR et la molécule thérapeutique comprenant un agent et l'anticorps à domaine unique. L'invention concerne également un procédé de transport d'une telle molécule thérapeutique vers la circulation systémique ou le chorion ou le tractus gastro-intestinal ou le tractus gastro-intestinal d'un sujet, comprenant l'administration de la molécule thérapeutique au sujet par administration orale, buccale, nasale ou par inhalation.
EP20849929.3A 2019-08-02 2020-07-31 Matières et procédés pour le biotransport multidirectionnel Pending EP4007591A4 (fr)

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
US201962882387P 2019-08-02 2019-08-02
US201962882291P 2019-08-02 2019-08-02
US201962882346P 2019-08-02 2019-08-02
US201962882361P 2019-08-02 2019-08-02
US201962940228P 2019-11-25 2019-11-25
US201962940200P 2019-11-25 2019-11-25
US201962940232P 2019-11-25 2019-11-25
US201962940220P 2019-11-25 2019-11-25
US201962940196P 2019-11-25 2019-11-25
US201962940206P 2019-11-25 2019-11-25
US201962940208P 2019-11-25 2019-11-25
PCT/US2020/044497 WO2021025997A1 (fr) 2019-08-02 2020-07-31 Matières et procédés pour le biotransport multidirectionnel

Publications (2)

Publication Number Publication Date
EP4007591A1 true EP4007591A1 (fr) 2022-06-08
EP4007591A4 EP4007591A4 (fr) 2023-11-29

Family

ID=74502941

Family Applications (2)

Application Number Title Priority Date Filing Date
EP20849929.3A Pending EP4007591A4 (fr) 2019-08-02 2020-07-31 Matières et procédés pour le biotransport multidirectionnel
EP20850058.7A Pending EP4007607A4 (fr) 2019-08-02 2020-07-31 Matériaux et procédés de ciblage d'un récepteur d'anticorps polymère

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP20850058.7A Pending EP4007607A4 (fr) 2019-08-02 2020-07-31 Matériaux et procédés de ciblage d'un récepteur d'anticorps polymère

Country Status (18)

Country Link
US (2) US20230019640A1 (fr)
EP (2) EP4007591A4 (fr)
JP (2) JP2022542391A (fr)
KR (2) KR20220054585A (fr)
CN (2) CN114173801A (fr)
AU (2) AU2020326590A1 (fr)
BR (2) BR112022001352A2 (fr)
CA (2) CA3147905A1 (fr)
CO (2) CO2022000817A2 (fr)
CR (2) CR20220043A (fr)
DO (2) DOP2022000021A (fr)
EC (2) ECSP22007690A (fr)
IL (2) IL289955A (fr)
JO (1) JOP20220025A1 (fr)
MX (2) MX2022001379A (fr)
PE (2) PE20220298A1 (fr)
TW (2) TW202116813A (fr)
WO (2) WO2021025997A1 (fr)

Family Cites Families (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1221428A (zh) * 1996-06-04 1999-06-30 加利福尼亚大学董事会 Pigr生物大分子特征性高级结构和相关配体的细胞内化作用
US6060082A (en) * 1997-04-18 2000-05-09 Massachusetts Institute Of Technology Polymerized liposomes targeted to M cells and useful for oral or mucosal drug delivery
CA2362373A1 (fr) * 1999-02-12 2000-08-17 J. Donald Capra Domaines se liant a un recepteur polymere d'immunoglobuline (pigr), et leurs techniques d'utilisation
MXPA02009449A (es) * 2000-03-27 2003-09-22 Univ California Ligandos dirigidos a la region no del eje del componente no secretor de la pigr y metodos de uso de los mismos.
US20030166160A1 (en) * 2001-09-06 2003-09-04 Hawley Stephen B. Compounds and molecular complexes comprising multiple binding regions directed to transcytotic ligands
IL155204A0 (en) * 2000-10-02 2003-11-23 Arizeke Pharmaceuticals Inc Compositions and methods for the transport of biologically active agents across cellular barriers
WO2002083840A2 (fr) * 2001-04-03 2002-10-24 Arizeke Pharmaceuticals, Inc. Compositions et methodes de transport transepithelial de vesicules membranaires et de virions
WO2004062602A2 (fr) * 2003-01-09 2004-07-29 Arizeke Pharmaceuticals Inc. Compositions et methodes de transport biologique cible de supports moleculaires
WO2008074868A1 (fr) * 2006-12-20 2008-06-26 Ablynx N.V. Administration par voie orale de polypeptides
US20100166734A1 (en) * 2006-12-20 2010-07-01 Edward Dolk Oral delivery of polypeptides
EP1975178A1 (fr) * 2007-03-30 2008-10-01 f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H. Anticorps modulaire transcytotique
WO2009080764A2 (fr) * 2007-12-20 2009-07-02 Abylnx N.V. Administration orale ou nasale de composés comprenant des séquences d'acides aminés
WO2013036130A1 (fr) * 2011-09-09 2013-03-14 Universiteit Utrecht Holding B.V. Vhh à neutralisation large dirigés contre le vih-1
EP2807189B1 (fr) * 2012-01-23 2019-03-20 Ablynx N.V. Séquences dirigées contre le facteur de croissance des hépatocytes (hgf) et polypeptides les comprenant pour le traitement de cancers et/ou de tumeurs
CN104903727B (zh) * 2012-11-08 2019-10-22 马克法兰伯尼特医学研究与公共健康协会有限公司 诊断、预后、治疗和筛选方案
EP3436475A1 (fr) * 2016-03-29 2019-02-06 STCube & Co., Inc. Procédés de sélection d'anticorps qui se lient spécifiquement à des protéines de point de contrôle immunitaire glycosylées
TW201900213A (zh) * 2017-05-27 2019-01-01 美商免疫醫療公司 免疫球蛋白a陽性細胞的調節

Also Published As

Publication number Publication date
JP2022542391A (ja) 2022-10-03
DOP2022000022A (es) 2022-07-31
BR112022001080A2 (pt) 2022-07-19
EP4007607A1 (fr) 2022-06-08
ECSP22014913A (es) 2022-03-31
PE20220298A1 (es) 2022-03-07
BR112022001352A2 (pt) 2022-06-07
US20220324970A1 (en) 2022-10-13
IL289955A (en) 2022-03-01
WO2021026000A1 (fr) 2021-02-11
CR20220043A (es) 2022-06-06
PE20220344A1 (es) 2022-03-14
TW202122108A (zh) 2021-06-16
AU2020326590A1 (en) 2022-02-17
CN114173801A (zh) 2022-03-11
CA3147905A1 (fr) 2021-02-11
CA3147916A1 (fr) 2021-02-11
EP4007591A4 (fr) 2023-11-29
AU2020326589A1 (en) 2022-02-17
US20230019640A1 (en) 2023-01-19
DOP2022000021A (es) 2022-08-15
MX2022001379A (es) 2022-03-17
KR20220054585A (ko) 2022-05-03
CN114423451A (zh) 2022-04-29
JOP20220025A1 (ar) 2023-01-30
CR20220042A (es) 2022-03-22
MX2022001278A (es) 2022-05-03
WO2021025997A1 (fr) 2021-02-11
IL289956A (en) 2022-03-01
KR20220054289A (ko) 2022-05-02
EP4007607A4 (fr) 2024-01-10
JP2022542418A (ja) 2022-10-03
TW202116813A (zh) 2021-05-01
ECSP22007690A (es) 2022-02-25
WO2021026000A8 (fr) 2022-02-17
CO2022000817A2 (es) 2022-02-07
CO2022001579A2 (es) 2022-03-18

Similar Documents

Publication Publication Date Title
US20210340272A1 (en) Anti-mesothelin constructs and uses thereof
US20210380675A1 (en) Il-36 antibodies and uses thereof
US10836833B2 (en) Cell engaging binding molecules
US20220112276A1 (en) Biosynthetic materials and methods for multidirectional biotransportation
US20220289857A1 (en) Fn14 antibodies and uses thereof
US20240182582A1 (en) Materials and methods for immune effector cells redirection
US20220324970A1 (en) Materials and methods for multidirectional biotransportation
US20220267456A1 (en) Materials and methods for targeting regulatory t cells for enhancing immune surveillance
WO2022161472A1 (fr) Molécules de liaison du sars-cov-2 et leurs utilisations
US20230227545A1 (en) Materials and methods of il-1beta binding proteins
WO2024013727A1 (fr) Matériau et procédés d'appariement amélioré par génie biologique de régions variables de liaison à l'antigène

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20220131

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40073846

Country of ref document: HK

A4 Supplementary search report drawn up and despatched

Effective date: 20231026

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 1/14 20060101ALI20231020BHEP

Ipc: A61P 1/00 20060101ALI20231020BHEP

Ipc: A61K 45/00 20060101ALI20231020BHEP

Ipc: A61K 38/00 20060101AFI20231020BHEP